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WO2022224035A2 - Anti-gm1 antibody binding compounds - Google Patents

Anti-gm1 antibody binding compounds Download PDF

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Publication number
WO2022224035A2
WO2022224035A2 PCT/IB2022/000224 IB2022000224W WO2022224035A2 WO 2022224035 A2 WO2022224035 A2 WO 2022224035A2 IB 2022000224 W IB2022000224 W IB 2022000224W WO 2022224035 A2 WO2022224035 A2 WO 2022224035A2
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WIPO (PCT)
Prior art keywords
compound
optionally substituted
amino acid
polymer
gml
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PCT/IB2022/000224
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French (fr)
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WO2022224035A3 (en
Inventor
Jérémy BOILEVIN
Lijuan PANG
Pascal HÄNGGI
Ruben HERRENDORFF
Beat Ernst
Butrint Aliu
Horst Prescher
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Polyneuron Pharmaceuticals Ag
Universität Basel
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Publication of WO2022224035A2 publication Critical patent/WO2022224035A2/en
Publication of WO2022224035A3 publication Critical patent/WO2022224035A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the invention relates to compounds including carbohydrate ligands that bind to antibodies (IgG and IgM isotype) against the GM1 ganglioside, and to their use in diagnostic and therapeutic applications related to anti-GMl antibody-associated neuropathies, such as Guillain-Barre-Syndrome (GBS) and multifocal motor neuropathy (MMN).
  • GBS Guillain-Barre-Syndrome
  • MNN multifocal motor neuropathy
  • Anti -ganglioside antibodies particularly anti-GMl antibodies have been detected in a various peripheral neuropathies, including variants of GBS such as acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) and the pharyngeal -cervical-brachial variant of GBS, and the chronic multifocal motor neuropathy MMN.
  • GBS has an incidence of approximately 1-2 in 100,000 people whereas the prevalence of MNN is approximately 1 in 100,000 people.
  • GM1 belongs to the family of gangliosides (glycosphingolipids), it is composed of a ceramide tail and a pentasaccharide head group containing one sialic acid (N-acetylneuraminic acid, Neu5Ac). GM1 occurs widely in human tissues, where it exhibits a variety of essential functions, both in the plasma membrane and intracellular loci. GM1 is typically located in membrane rafts and functionally prominent in lipid microdomains.
  • GM1 interacts with proteins that modulate mechanisms such as ion transport (transport of Ca 2+ , Na + , and K + via ion channels and ion exchangers), neuronal differentiation, signaling via G protein-coupled receptors (GPCRs), immune system reactivities (effector T cells), and neuroprotective signaling.
  • GPCRs G protein-coupled receptors
  • effector T cells effector T cells
  • neuroprotective signaling occurs through intimate association with neurotrophin receptors, which has relevance to the etiopathogenesis of neurodegenerative diseases and potential therapies.
  • This disclosure provides glycan-conjugated compounds that specifically bind to anti-GMl autoantibodies.
  • These glycan-conjugated compounds can be conjugated to a polymeric backbone and/or support configured to display multiple glycan groups and designed to mimic the natural GM1 epitope.
  • the glycan-conjugated compounds can also be configured on a polymeric support such as a Sepharose/agarose bead suitable for purification or separation of biological samples.
  • the linked glycan groups are selected from a series of novel analogues of the GM1 epitope (FIG. 1).
  • glycan-containing compounds of formula (I) that mimic the natural GM1 epitope, and therapeutically useful polymers that include a multitude of such ligands, designed to bind anti-GMl antibodies in vitro for diagnostic use, and more importantly designed to sequester and eliminate anti-GMl antibodies in vivo or extracorporeal (ex vivo) for the treatment anti-GMl autoantibody related neuropathies, such as AMAN, AMSAN, and MMN.
  • FIG. 1 Representative structures of the natural GM1 epitope (compound 1) and a series of GM1 glyco-mimetics with modifications on the GM1 core structure, such as: a) replacement of the reducing end glucose (Part-I) with a tyramine moiety (compound 2-7); b) replacement of the Neu5Ac (Part-II) with 2-(3 -cyclohexyl -propanoic acid), 2-(3 -phenyl-propanoic acid), and 2-propanoic acid (compound 3-9).
  • FIGs. 2A-2C Representative retrosynthesis of the exemplary GM1 ligand compounds including exemplary GM1 glyco-mimetics.
  • Sialic acid is first attached to the 3 -position of galactosides (23a-b) and the propionates are introduced via tin-mediated alkylation reactions (®29a- c, 34a-b, 37a-b) (FIG. 2C).
  • Two different Gal-GalN building blocks 13, 17 are prepared from monosaccharide donors (10, 14) and acceptors (11, 15) and used in the subsequent glycosylation reactions (FIG. 2B).
  • the key step of the GM1 synthesis is the coupling of Gal-GalN disaccharide fragments (13, 17) to selectively protected di- or tri-saccharide acceptors (29a-c, 34a-b, 37a-b) via both the conventional and pre -activation glycosylation conditions (FIG. 2A).
  • FIGs. 3A-3B show the inhibitory activity of exemplary polymeric compounds.
  • FIG. 3A shows inhibition of binding of selected MMN patients’ sera anti-GMl IgM to GM1 ganglioside by exemplary polymeric compounds 59-68.
  • FIGs. 3C-3D shows the depletion of anti-GMl IgG (FIG. 3C) and anti-GMl IgM (FIG. 3D) antibodies from patient serum using GM1 mimetic (2)-functionalized Sepharose beads.
  • FIG. 4 shows the effects of temperature on the inhibitory activity of exemplary polymeric compounds. Temperature dependent inhibition of anti-GMl IgG to the GM1 ganglioside by polymer 62 and temperature independent inhibition by polymers 60 and 68 (***p> 0.001).
  • FIG. 5 Ex vivo inhibition of anti-GMl IgG binding and complement deposition to murine nerve terminals by polymer 60 (***p> 0.001).
  • FIG. 6A-6C shows inhibition of anti-GMl IgG binding to phrenic nerves by exemplary polymeric compound in an in vivo nerve injury mouse model of AMAN.
  • FIG. 7A depicts sections of murine diaphragm used for in vitro investigation.
  • FIG. 7B illustrates triangularis stemi nerve-muscle preparations, dissected out of mouse rib cage, used for ex vivo investigation (created using BioRender).
  • FIG. 8A shows a graph of in vitro dose response for binding and sequestering anti-GMl antibodies by an exemplary polymeric compound as compared to a control polymeric compound (control mimetic) that does not bind anti- GMl antibody.
  • FIG. 8B shows the related images of diaphragm tissue from neuronal GM1 -enriched mice.
  • Btx nicotinic acetylcholine receptor;
  • DG2 anti-GMl IgG antibody.
  • Scale Bar 50 pm.***p ⁇ 0.001, **p ⁇ 0.01, one-way ANOVA.
  • FIG. 9A shows a graph of DG2 antibody staining observed in the triangularis stemi nerve-muscle tissue for exemplary polymeric compound as compared to control polymeric compound (control mimetic) that does not bind anti-GMl antibody.
  • FIG. 9B shows related images of tissue preparations from neuronal GM1 -enriched mice stained with bungarotoxin to visualize the nerve-terminal and an anti-mouse IgG3 antibody to measure DG2 antibody.
  • Btx nicotinic acetylcholine receptor
  • DG2 anti-GMl IgG antibody.
  • Scale Bar 20 pm;****p ⁇ 0.0001, unpaired two-tailed student t-test. 5.
  • the compounds and conjugates include several glycan groups linked to a moiety of interest (e.g., a polymer, carrier or support).
  • the compounds include one or more glycan groups linked to a chemoselective ligation group, or a synthetic precursor thereof, suitable for use in preparation of a compound or conjugate including a linked moiety of interest.
  • the glycan groups are designed to mimic the pentasaccharide head group of GM1 and incorporate an aryl or heteroaryl linking moiety that provides connection via a linker to the moiety of interest with a configuration that provides potent binding to the anti-GMl autoantibodies, especially anti-GMl autoantibodies having neuropathic potential.
  • glycan group-containing compounds can provide potent binding and blocking of the disease-causing target anti-GMl autoantibodies.
  • constructs can be used as a basis for multivalent display of the glycan groups to provide for desirable binding and blocking activity against target anti-GMl autoantibodies, and are meant to be encompassed by the compounds and conjugates of this disclosure.
  • the construct is a polymer that provides multiple points of attachment for the glycan groups.
  • the construct is a support, such as a bead or planar solid support suitable for multivalent display of the glycan groups.
  • the particular constructs, compounds and conjugates of this disclosure can be selected to provide desirable binding and blocking activity against target anti-GMl autoantibodies depending on the particular biological sample or system in which it is used.
  • exemplary anti-GMl antibody-binding polymeric compounds and conjugates of this disclosure bind and block target anti-GMl antibodies in a biological system, e.g., the human serum of a patient (ex vivo) or in vivo in a mouse model for AMAN. Accordingly, the anti- GMl antibody-binding compounds and conjugates of this disclosure are useful in anti-GMl antibody associated neuropathy applications, including therapeutic and diagnostic applications.
  • the anti-GMl antibody-binding compounds of this disclosure can include one or more, two or more, or several glycan groups that mimic GM1, and which can be linked via a chemoselective ligation group to a moiety of interest. It is understood that the anti-GMl antibody-binding compounds can be conjugated to moiety of interest suitable for an in vivo, or ex vivo therapeutic or diagnostic use.
  • GM1 is a glycosphingolipid that belongs to the family of gangliosides and is composed of a ceramide tail and a penta-saccharide head group containing the sialic acid, N-acetylneuraminic acid (Neu5Ac).
  • Neuropathy indications such as AMAN, AMSAN, and MMN.
  • the glycan group has a tetra-saccharide portion of the head group that includes a sialic acid group.
  • the sialic acid group is the naturally occurring N- acetylneuraminic acid, or an analog such as N-g 1 y co hi n c uram i n i c acid.
  • glycan groups with a modified tri-saccharide having a suitable sialic acid group replacement that is an optionally substituted carboxymethyl group (e.g., a HO 2 C-CH 2 - group where the methylene carbon is further substituted) can provide for desirable high affinity binding to target anti-GMl autoantibodies.
  • the glycan group of the anti-GMl antibody-binding compound is of formula (XI): wherein:
  • R 1 is a sialic acid group or an optionally substituted carboxymethyl group
  • Z 1 is -0-, -S-, -NR 2 - or -C(R 2 ) 2 -, wherein each R 2 is independently selected from H, (C1-C4)- alkyl, (Ci-C 4 )-alkoxy, -CH 2 C 6 H 5 , -CH 2 CH 2 C6H5, -OCH 2 C 6 H 5 , and -OCH 2 CH 2 C6H5;
  • Ar is an optionally substituted aryl or optionally substituted heteroaryl; and L 1 is a linker (e.g., as described herein).
  • Ar is optionally substituted aryl or optionally substituted heteroaryl.
  • Ar is an optionally substituted 5-membered monocyclic heteroaryl group. In some embodiments of formula (XI), Ar is an optionally substituted 6- membered monocyclic aryl or heteroaryl group.
  • the Ar group linking moiety of formula (XI) can be an optionally substituted multicyclic aryl or multicyclic heteroaryl group, such as an optionally substituted bicyclic aryl or bicyclic heteroaryl group.
  • Ar is an optionally substituted fused bicyclic group.
  • Ar is an optionally substituted bicyclic group comprising two aryl and/or heteroaryl monocyclic rings connected via a covalent bond.
  • Ar is an optionally substituted bicyclic aryl or bicyclic heteroaryl group having two 6-membered rings. In some embodiments of formula (XI), Ar is an optionally substituted bicyclic aryl or bicyclic heteroaryl group having one 6-membered ring that is connected via a covalent bond or fused to a 5-membered ring.
  • each Ar is independently selected from optionally substituted phenyl, optionally substituted pyridyl, optionally substituted biphenyl, optionally substituted naphthalene, and optionally substituted quinoline.
  • Ar is substituted with one or substituents selected from OH, halogen, (Ci-Cg)-alkyl, optionally substituted (Ci-Cg)-alkyl, (Ci-Cg)-alkoxy, and optionally substituted (Ci-Cg)-alkoxy.
  • Ar is optionally substituted 1,4-phenylene, optionally substituted 1,3- phenylene, or optionally substituted 2,5-pyridylene.
  • the glycan group of the anti-GMl antibody-binding compound is of formula (XII): or a pharmaceutically acceptable salt thereof, wherein: q is 0 to 4; and each R 11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
  • R 1 is a sialic acid group.
  • Sialic acid group refers to a monosaccharide of the sialic acid class of alpha-keto acid sugars having a nine-carbon backbone.
  • the sialic acid can be naturally occurring, e.g., as terminating branches of N-glycans, O-glycans, and glycosphingolipids (gangliosides).
  • the sialic acid group is a N- and O- substituted derivative of neuraminic acid.
  • the sialic acid group is N- acetylneuraminic acid.
  • the sialic acid group is N- glycolylneuraminic acid.
  • R 1 is optionally substituted carboxymethyl group, e.g., a HO2C-CH2- group wherein the methylene carbon is further optionally substituted.
  • R 1 is selected from formula (XHIa) and (Xlllb):
  • R 21 is optionally substituted (Ci-C3)-alkyl
  • R 22 and R 23 are independently selected from H, optionally substituted (Ci-C3)-alkyl, optionally substituted aryl-(Ci-C3)-alkylene-, optionally substituted heteroaryl-(Ci-C3)-alkylene-, optionally substituted cycloalkyl-(Ci-C3)-alkylene-, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl.
  • R 21 is methyl. In some embodiments of formula (XHIa), R 21 is hydroxymethyl (HO-CH2-).
  • R 1 is of formula (XHIa), wherein R 21 is methyl: or a pharmaceutically acceptable salt thereof.
  • R 1 is of formula (XHIa), wherein R 21 is hydroxymethyl: or a pharmaceutically acceptable salt thereof.
  • R 22 is H and R 23 is not H.
  • R 22 is H and R 23 is optionally substituted (C1-C3)- alkyl. In some embodiments of formula (XHIb), R 22 is H and R 23 is optionally substituted aryl-(Ci- C3)-alkylene-. In some embodiments of formula (XHIb), R 22 is H and R 23 is optionally substituted phenyl-(Ci-C3)-alkylene-. In some embodiments of formula (XHIb), R 22 is H and R 23 is optionally substituted benzyl. In some embodiments of formula (XHIb), R 22 is H and R 23 is optionally substituted cyclohexyl-(Ci-C3)-alkylene-. In some embodiments of formula (XHIb), R 22 is H and R 23 is optionally substituted cyclohexyl-methyl-.
  • R 1 is of (XHIb) and selected from: or a pharmaceutically acceptable salt thereof.
  • R 1 is selected from: or a pharmaceutically acceptable salt thereof.
  • Z 1 is -0-. In some embodiments of formula (XI)-(XII), Z 1 is -S-.
  • Z 1 is -NR 2 -, where R 2 is selected from H, (Ci- C 4 ) -alkyl, (Ci-C 4 )-alkoxy, -CH 2 C 6 H 5 , -CH 2 CH 2 C 6 H 5 , -OCH 2 C 6 H 5 , and -OCH 2 CH 2 C 6 H 5 .
  • Z 1 is -NR 2 -, where R 2 is selected from H, and (Ci-C 4 )-alkyl.
  • Z 1 is -NMe-.
  • Z 1 is -NH-.
  • Z 1 is -C(R 2 ) 2 -. In some embodiments of formula (XI)-(XII), Z 1 is -CH 2 -.
  • Exemplary glycan groups include, but are not limited to, PCT/IB2022/000224
  • the linked glycan is or a pharmaceutically acceptable salt thereof.
  • the linked glycan is or a pharmaceutically acceptable salt thereof.
  • the linked glycan is or a pharmaceutically acceptable salt thereof.
  • linker (L 1 ) can be incorporated into the glycans of formula (XI)-(XII) to connect the glycan group of this disclosure to a chemoselective ligation group or other moiety of interest (e.g., Y of formula (I), or P of formula (III), or Z 2 of formula (V), as described herein).
  • linker “linking moiety” and “linking group” are used interchangeably and refer to a linking moiety that covalently connects two or more moieties or compounds, such as glycans and other moieties of interest.
  • the linker is divalent and connects a single glycan group to a moiety of interest.
  • the linker is a branched linking group that is trivalent or of a higher multivalency, and is capable of linking two, three or more glycan groups to a moiety of interest.
  • the linker that connects the two or more moieties has a linear or branched backbone of 500 atoms or less (such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less) in length, e.g., as measured between the two or more moieties.
  • 500 atoms or less such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less
  • a linking moiety may be a covalent bond that connects two groups or a linear or branched chain of between 1 and 500 atoms in length, for example of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, 200, 300, 400 or 500 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom. In certain cases, one, two, three, four, five or more, ten or more, or even more carbon atoms of a linker backbone may be optionally substituted with heteroatoms, e.g., sulfur, nitrogen or oxygen heteroatom.
  • heteroatoms e.g., sulfur, nitrogen or oxygen heteroatom.
  • linker when the linker includes an ethylene glycol or polyethylene glycol) group, every third atom of that segment of the linker hydrocarbon backbone is substituted with an oxygen.
  • bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
  • the linker may include one or more substituent groups.
  • a linker may further include, without limitations, one or more of the following: ether, thioether, disulfide, amide, carbonate, carbamate, tertiary amine, or alkyl, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), nbutyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like.
  • the linker backbone may include a cyclic group, for example, an aryl, a heterocycle, a cycloalkyl group or a heterocycle group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
  • a “linker” or linking moiety, or a portion thereof is derived from a molecule with two reactive termini (e.g., a bifunctional linker), one for conjugation to a moiety of interest (e.g., as described herein) and the other for linkage to a glycan group.
  • a bifunctional linker e.g., one for conjugation to a moiety of interest (e.g., as described herein) and the other for linkage to a glycan group.
  • the linker L 1 includes one or more straight or branched-chain carbon moieties and/or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CH2CH2O- or -OCH2CH2-), and combinations thereof, optionally connected via one or more linkages.
  • these linkers optionally have amide linkages, urea or thiourea linkages, carbamate linkages, ester linkages, amino linkages, ether linkages, thioether linkages, sulfhydryl linkages, or other hetero-functional linkages.
  • the linker comprises one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
  • the linker comprises one or more of an ether bond, thioether bond, amine bond, amide bond, carbon-carbon bond, carbon- nitrogen bond, carbon-oxygen bond, carbon-sulfur bond, and combinations thereof.
  • the linker comprises a linear structure.
  • the linker comprises a branched structure.
  • the linker comprises a cyclic structure.
  • L is a linker between about 5 A and about 500 A. In certain embodiments, L is between about 10 A and about 400 A. In certain embodiments, L is between about 10 A and about 300 A. In certain embodiments, L is between about 10 A and about 200 A. In certain embodiments, L is between about 10 A and about 100 A.
  • the linker may be considered as connecting directly to the Ar group of a glycan compound (e.g., Ar of Formula (XI)-(XII) as described herein). It is understood that the linker may encompass one or more linking functional groups that are the residual product of a coupling reaction between two chemoselective ligation groups (e.g., as described herein). Residual linking functional groups derived from any of the conjugation chemistries described herein can be incorporated into the linkers of the compounds and conjugates of this disclosure.
  • L 1 is linear linker comprising one or more linking moieties independently selected from -Ci- 6 -alkylene-, -NHCO-Ci- 6 -alkylene-, -CONH-C1-6- alkylene-, -NH Ci- 6 -alkylene-, -NHCONH-Ci- 6 -alkylene-, - NHCSNH-Ci- 6 -alkylene-, -C1-6- alkylene-NHCO, -Ci- 6 -alkylene-CONH-, -Ci- 6 -alkylene-NH-, -Ci- 6 -alkylene-NHCONH-, -C1-6- alkylene-NHC SNH-, -0(CH 2 ) P- , -(OCH 2 CH 2 ) P- , -NHCO-, -CONH-, -NHSO2-, -SO2NH-,
  • the glycan groups of this disclosure can be linked to a moiety of interest (Y) via a linker (L 1 ).
  • the linkage of a glycan-linker compound to a moiety of interest (Y) is achieved via a conjugation or ligation reaction between two compatible functional groups, i.e., a first chemoselective ligation group that is incorporated into the linker of a glycan-linker compound, and a compatible chemoselective ligation group of the moiety of interest.
  • a variety of chemoselective ligation groups, or synthetic precursors thereof, can be incorporated into the glycan-linker compounds of this disclosure.
  • a chemoselective ligation group is a group having a reactive functionality or functional group capable of conjugation to a compatible group of a second moiety.
  • chemoselective ligation groups may be one of a pair of groups associated with a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Pictet- Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g., thiol-maleimide, thiol- haloacetamide or alkyne hydrothiolation), amine-active ester amido bond coupling, reductive amination, dialkyl squarate chemistry, etc.
  • a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Pictet- Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g
  • Chemoselective ligation groups that may be utilized in linking a glycan-linker to a moiety of interest, include, but are not limited to, amine (e.g., a N-terminal amine or a lysine side chain amine group of a polypeptide), azide, aryl azide, alkynyl (e.g., ethynyl or cyclooctyne or derivative), active ester (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester or pentafluorophenyl (PFP) ester or thioester), haloacetamide (e.g., chloroacetamide, iodoacetamide or bromoacetamide), chloroacetyl, bromoacetyl, hydrazide, maleimide, vinyl sulfone, 2-sulfonyl pyridine, cyan
  • a chemoselective ligation group is capable of spontaneous conjugation to a compatible chemical group when the two groups come into contact under suitable conditions (e.g., copper free Click chemistry conditions). In some instances, the chemoselective ligation group is capable of conjugation to a compatible chemical group when the two groups come into contact in the presence of a catalyst or other reagent (e.g., copper catalyzed Click chemistry conditions).
  • suitable conditions e.g., copper free Click chemistry conditions
  • the chemoselective ligation group is capable of conjugation to a compatible chemical group when the two groups come into contact in the presence of a catalyst or other reagent (e.g., copper catalyzed Click chemistry conditions).
  • the chemoselective ligation group is a photoactive ligation group.
  • a diazirine group upon irradiation with ultraviolet light, can form reactive carbenes, which can insert into C-H, N-H, and O-H bonds of a second moiety.
  • Z 2 is a precursor of the reactive functionality or function group capable of conjugation to a compatible group of a second moiety.
  • a carboxylic acid is a precursor of an active ester chemoselective ligation group.
  • Z 2 is a reactive moiety capable forming a covalent bond to a polypeptide (e.g., with an amino acid side chain of a polypeptide having a compatible reactive group).
  • the reactive moiety can be referred to as a chemoselective ligation group.
  • Z 2 is a thiol-reactive chemoselective ligation group.
  • Z 2 can produce a residual moiety (Z 3 ) that results from the covalent linkage of a thiol with a thiol-reactive chemoselective ligation group of a polymer backbone residue, e.g., a polypeptide having side chain groups that are modified to include a thiol-reactive chemoselective ligation group, such as a haloacetamide.
  • Z 2 is an amine group, that can be coupled with an amino-reactive chemoselective ligation group of a moiety of interest.
  • Z 2 can produce a residual moiety (Z 3 ) resulting from the covalent linkage of an amine and an amine-reactive chemoselective ligation group (e.g., an active ester group) of a moiety of interest, e.g., Z 3 is an amide linkage.
  • the glycan groups of this disclosure can be linked to a moiety of interest (Y) via a linker (L 1 ).
  • a linker L 1
  • another aspect of this disclosure is glycan-linker compounds that include a chemoselective ligation group useful in attaching the linked glycans to a variety of constructs for the preparation of compounds and conjugates of this disclosure.
  • the glycan-linker compound includes a glycan of formula (XI)-(XII), and is of formula (V): or a salt thereof, wherein:
  • R 1 is a sialic acid group or an optionally substituted carboxymethyl group.
  • Z 1 is -0-, -S-, -NR 2 - or -C(R 2 )2-, wherein each R 2 is independently selected from H, (C1-C4)- alkyl, (Ci-C 4 )-alkoxy, -CH 2 C 6 H 5 , -CH 2 CH 2 C6H 5 , -OCH 2 C 6 H 5 , and -OCH 2 CH 2 C 6 H 5 ;
  • Ar is optionally substituted aryl or optionally substituted heteroaryl (e.g., monocyclic aryl or heteroaryl, or bicyclic aryl or heteroaryl);
  • L 1 is a linker
  • Z 2 is a chemoselective ligation group, or a precursor thereof.
  • L 1 is linear linker comprising one or more linking moieties independently selected from -Ci- 6 -alkylene-, -NHCO-Ci- 6 -alkylene-, -CONH-C1-6- alkylene-, -NH Ci- 6 -alkylene-, -NHCONH-Ci- 6 -alkylene-, - NHCSNH-Ci- 6 -alkylene-, -C1-6- alkylene-NHCO, -Ci- 6 -alkylene-CONH-, -Ci- 6 -alkylene-NH-, -Ci- 6 -alkylene-NHCONH-, -C1-6- alkylene-NHC SNH-, -0(CH 2 ) p- - (OCH 2 CH 2 ) p- - NHCO-, -CONH-, -NHS0 2- , -S0 2 NH-, -CO-,
  • Z 2 is selected from -NH 2 , -SH, -N3, alkyne, active ester, and maleimide.
  • Z 2 is amine (e.g., -NH 2 ).
  • Z 2 is thiol (e.g., -SH).
  • -L'-Z 2 is -(Ci-C 6 )-alkyl-NH 2 .
  • -L'-Z 2 is -(CH 2 ) 2 NH 2 .
  • the linker of the compound can be extended to incorporate a longer L 1 and/or an alternative Z 2 .
  • a bifunctional linker or reagent can be attached to the terminal Z 2 chemoselective ligation group, to produce another glycan-linker compound of formula (V).
  • Exemplary bifunctional linkers or reagents which can be utilized to extend or modify the glycan-linker compounds of formula (V) include, but are not limited to, Traut’s reagent and g-thiobutyrolactone.
  • -L'-Z 2 is -(Ci-C 6 )-alkyl-amido-(Ci-C 6 )-alkyl-SH. In some embodiments of formula (V), -L'-Z 2 is -(CH 2 ) 2 NHCO(CH 2 )3SH.
  • the compound is selected from:
  • -L 1 - is -Z 11 -(Ci-C 6 )-alkyl-Z 12 -(Ci-C 6 )-alkyl-;
  • Z 2 is NH 2 .
  • Z 2 is SH.
  • -L'-Z 2 is -(Ci-C 6 )-alkyl-amido-(Ci-C 6 )-alkyl-SH. In some embodiments of these compounds, -L'-Z 2 is -(CH2)2NHCO(CH2)3SH.
  • the compound is or a salt thereof.
  • the compound is or a salt thereof.
  • the compound is or a salt thereof.
  • the compound is or a salt thereof.
  • the compound is or a salt thereof. [0084] In some embodiments of formula (V), the compound is or a salt thereof.
  • aspects of this disclosure include glycan-containing compounds and conjugates that include a glycan group (e.g., of formula (XI)-(XII) described herein) linked to a moiety of interest.
  • a glycan group e.g., of formula (XI)-(XII) described herein
  • the linkage of glycan-linker compounds to a moiety of interest (Y) can be achieved via a chemoselective ligation (e.g., as described herein).
  • the compounds of this disclosure can be referred to interchangeably as conjugates, e.g., when the moiety of interest (Y) is a molecule or construct such as, a polymer, carrier biomolecule or support.
  • conjugates can be prepared by coupling of a chemoselective ligation group on a glycan- linker compound with a compatible reactive group of Y.
  • the compatible reactive group can be introduced into a moiety of interest by modification prior to conjugation, or can be a group already present in Y.
  • the conjugates of this disclosure can be prepared by incorporating a linker into a moiety of interest to which a glycan group can be attached.
  • the moiety of interest Y is a polymer.
  • a variety of polymers can be utilized that have a polymer backbone composed of repeat units providing for attachment of a multitude of linked glycan compounds.
  • the polymer can be a homopolymer.
  • the polymer can be a heteropolymer, such as a block copolymer or a random copolymer.
  • the polymer can be prepared in a stepwise fashion and have a discrete or defined length and sequence. Alternatively, the polymer can be prepared via a bulk polymerization method.
  • the polymer is pharmaceutically acceptable, e.g., a biocompatible polymer suitable for therapeutic use in a polymeric compound of this disclosure.
  • the polymer is one suitable for in vivo applications where the polymer-glycan conjugate can be administered as a therapeutic agent to a subject in need thereof.
  • the polymer is one suitable for ex vivo applications, e.g., where the polymer-glycan conjugate can be used extracorporeally as part of an immune -adsorption support or apparatus configured to remove target autoantibodies from a biological sample.
  • Polymers of interest include, but are not limited to, amino acid polymers (referred to interchangeably as polypeptides), acrylic acid or methacrylic acid polymers or copolymers (e.g., polyacrylate, poly(meth)acrylate or copolymer thereof), N-vinyl-2-pyrrolidone-vinylalcohol copolymers, polysaccharide polymers, agarose, cellulose, chitosan polymers, and polyphosphazene polymers.
  • amino acid polymers referred to interchangeably as polypeptides
  • acrylic acid or methacrylic acid polymers or copolymers e.g., polyacrylate, poly(meth)acrylate or copolymer thereof
  • N-vinyl-2-pyrrolidone-vinylalcohol copolymers e.g., polysaccharide polymers, agarose, cellulose, chitosan polymers, and polyphosphazene polymers.
  • the polymer is an amino acid polymer.
  • the amino acid polymer can be composed of any convenient amino acid residues, including naturally occurring or non- naturally occurring amino acid residues, and including alpha- or beta- or delta- or gamma-amino acid residues, or any combinations thereof.
  • the polymer is an alpha-amino acid polymer.
  • Amino acids which can be incorporated into a polymer for use in the polymeric compounds of this disclosure include those having a side chain moiety suitable for conjugation to a glycan-linker compound (e.g., as described herein), such as amino acids selected from lysine, ornithine, glutamic acid, aspartic acid, serine and cysteine.
  • a side chain moiety suitable for conjugation to a glycan-linker compound e.g., as described herein
  • amino acids selected from lysine, ornithine, glutamic acid, aspartic acid, serine and cysteine amino acids selected from lysine, ornithine, glutamic acid, aspartic acid, serine and cysteine.
  • the side chain moiety of the amino acid can be modified (before or after polymerization) to incorporate a chemoselective ligation group.
  • the amino acid polymer includes residues that are selected to impart desirable properties (e.g., physical or optical properties such as hydrophilicity, hydrophobicity, charge, pi, UV absorbance, etc.) on the resulting polymeric compound.
  • desirable properties e.g., physical or optical properties such as hydrophilicity, hydrophobicity, charge, pi, UV absorbance, etc.
  • Amino acids of interest which may be incorporated into the polymer include, but are not limited to, lysine, ornithine, glutamic acid, aspartic acid, cysteine, alanine, glycine, serine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine, and the like.
  • the amino acid polymer can be linear, branched, hyperbranched or dendritic, as described by Z. Kadlecova et al, Biomacromolecules 2012, 13: 3127-3137.
  • the polymer backbone is an alpha-amino acid polymer, and the polymer is composed of amino acid resides selected from lysine, ornithine, glutamic acid, aspartic acid, and serine.
  • the polymer is poly-lysine. In some embodiments, the polymer is poly-Z -lysine. In some embodiments, the polymer is poly-D-lysine. In some embodiments, the polymer is a co-polymer of lysine residues and one or more additional amino acid residues (e.g., as described herein).
  • the polymer is poly-aspartic acid or poly-glutamic acid. In some embodiments, the polymer is poly-Z- aspartic acid. In some embodiments, the polymer is a co polymer of aspartic acid or glutamic acid residues and one or more additional amino acid residues (e.g., as described herein).
  • the side chains of a poly-aspartic acid or poly-glutamic acid polymer can be functionalized or derivatized by esterification and/or amide bond formation.
  • the molecular weight (MW) of the polymer backbone is lkDa to 300kDa, such as 10 to 200 kDa or 20 to 100 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is 25 to 85 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is 30 to 60 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is 40 to 60 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is 40 to 50 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is about 40 kDa.
  • the molecular weight (MW) of the polymer backbone is about 50 kDa. It is understood that the MW of the polymer backbone refers to the MW of the polymer without the glycan groups present, e.g., prior to conjugation of the glycan groups of formula (V).
  • the glycan groups that are attached to the polymer backbone are identical compounds of formula (XI), or include two or more different compounds formula (XI).
  • the polymer further comprises spacer moieties attached to side chain groups of the repeat units of the polymer to provide for linking to the glycan groups.
  • the polymer is an amino acid polymer, including lysine residues to which spacer moieties are attached, which spacer moieties themselves include reactive moieties (e.g., chemoselective ligation groups) that link to a compatible group of a gly can-linker compound (e.g., of formula (V), as described herein). Preferred examples are described herein.
  • the percentage of loading of the glycan group onto the polymer backbone is between 10 and 90%, preferably between 20 and 70%, between 20 and 60% or between 20 and 50%.
  • the loading refers to the % of repeat units of the polymer backbone that are linked to a glycan group (e.g., as described herein).
  • the loading refers to the % of amino acid residues of the polypeptide that are linked to a glycan group (e.g., as described herein).
  • the loading of the amino acid polymer is determined synthetically by controlling the equivalents of glycan-linker compounds of formula (V) that are coupled to the polymer backbone.
  • the loading of the amino acid polymer is determined by controlling the composition of the polypeptide backbone and limiting the number of residues that are capable of coupling with glycan-linker compounds of formula (V).
  • the percentage of loading of the glycan group onto the polymer backbone is typically and preferably determined by NMR spectroscopy and refers to % mole/mole.
  • the loading of a polymeric compound of this disclosure is determined using a quantitative NMR method based on a relative determination of integrals of interest.
  • the loading of a polymeric compound of this disclosure is determined using a quantitative NMR method based on an absolute concentration determination by comparing integrals of interest to standard.
  • the polymer is an amino acid polymer (also referred to as polypeptide)
  • amino acid polymer also referred to as polypeptide
  • some side chain residues of the polymer backbone that are not attached to glycan groups of this disclosure can be modified to incorporate a water solubilizing substituent.
  • the amino acid polymer includes lysine residues
  • some of the lysine residues may be capped with a water solubilizing substituent via amide bond formation, e.g., to an active ester group of a water solubilizing group precursor.
  • the polymer when the polymer is poly-lysine (e.g., as described herein) and a fraction of the lysine side chains are loaded with glycan groups, then substantially all of the remaining (uncapped) lysine side chains in the poly-lysine polymer are capped with a water solubilizing substituent.
  • water solubilizing substituent and “water soluble group” are used interchangeably and refer to a moiety, group or molecule that is hydrophilic and imparts improved water solubility upon a compound to which it is attached.
  • the water solubilizing substituent can be charged under physiological conditions, or can be a neutral hydrophilic group.
  • the water solubilizing substituent is a polyol group.
  • the water solubilizing substituent includes a (Ci-Cg)-alkyl substituted with one, two or more hydroxyl groups.
  • the water solubilizing substituent includes a dihydroxypropyl or dihydroxyethyl group.
  • a variety of chemoselective ligation groups and other linking groups or spacers can be utilized to link the water solubilizing substituent to the polymer.
  • the water solubilizing substituent is a group that can be charged under suitable aqueous conditions, e.g., a positively charged or negatively charged group, or a salt thereof, under physiological conditions.
  • the water solubilizing substituent is a carboxylic acid or a salt thereof.
  • the water solubilizing substituent is a sulfonic acid or a salt thereof.
  • the water solubilizing substituent is 2,3-dihydroxypropylthioacetyl.
  • the moiety of interest Y is a support.
  • supports may be utilized.
  • a support is composed of an inert material that is, or can be, functionalized to provide for attachment of a linked compound or moiety.
  • the support can be referred to as a solid support.
  • Supports of interest include, but are not limited to, beads, nanoparticles, planar supports, slides, microtiter plates (e.g., 96-well or other sized plates), membranes, monoliths, and the like.
  • the support is a bead.
  • utilization of monolithic solid-supports, membranes or planar solid supports can be advantageous.
  • the support e.g., beads
  • the support can be composed of a variety of materials, including but not limited to, agarose, Sepharose, polystyrene, divinylbenzene, silica, silica-based material, metal-based material, silicone, nitrocellulose, polymethacrylate, polyacrylate, and the like. Any immobilization chemistry can be selected as desired depending on the solid-support material.
  • the support is a bead suitable for ex vivo applications, e.g., where a bead immobilized-glycan can be used extracorporeally as part of an immune -adsorption support or apparatus configured to remove target autoantibodies from a biological sample.
  • Beads of interest include, but are not limited to, an agarose, a sepharose, a dextran, a cellulose, chitin, chitosan, derivatives thereof, an organic or inorganic porous material, a magnetic bead, or a micro bead.
  • the support is a bead, such as agarose beads or Sepharose beads (e.g., N-hydroxysuccinimide (NHS) activated Sepharose beads, or epoxy-activated Sepharose beads).
  • the beads are composed of paramagnetic core (e.g. Dynabeads).
  • the glycan-support e.g., gly can-bead, conjugates can be incorporated into an external immune-adsorption apparatus suitable for use in removing target autoantibodies from a biological sample, e.g., a blood, serum or plasma sample of a patient.
  • the beads can be utilized in a column or cartridge format, e.g., a format suitable for achieving separation of target anti-GMl autoantibodies from a biological sample.
  • the beads are suitable for chromatography.
  • the moiety of interest Y is a carrier molecule.
  • carrier and “carrier molecule” can refer to a biomolecule having multiple attachment points to which a glycan group can be linked.
  • the biomolecule can be one that is capable of transport of a linked compound through a biological system.
  • Y is a biomolecule.
  • Y is a biomolecule selected from protein, polynucleotide, polysaccharide, peptide, glycoprotein, lipid, enzyme, antibody, and antibody fragment.
  • Y is a carrier molecule
  • m is 2 to 50, such as 2 to 40, 2 to 30, 2 to 20 or 2 to 10.
  • a glycan-linker compound of formula (V) having a chemoselective ligation group can be conjugated to compatible amino acid side chain groups of a carrier protein.
  • the protein is itself modified to incorporate a chemoselective ligation group suitable for conjugation to a compatible glycan-linker compound of formula (V).
  • Z 2 is an amine-reactive group (e.g., an active ester, e.g., NHS ester) and can be used to conjugate one, two or more glycan groups to the protein, e.g., via conjugation to lysine sidechain residues of the protein.
  • Z 2 is a cysteine-reactive group (e.g., a maleimide) and can be used to conjugate one, two or more glycan groups to the protein.
  • Z 2 is a thiol (-SH) and can be used to conjugate one, two or more glycan groups to the protein, where the protein has been modified to incorporate a thiol-reactive group, such as a maleimide.
  • the ratio of glycan groups to carrier protein depends on the conjugation chemistry and stoichiometry of the ligation reaction.
  • m is an average number of glycan groups per protein, such as m is 2 to 50 glycan groups per protein, such as 2 to 40, 2 to 30, 2 to 20 or 2 to 10 glycan groups per protein.
  • the conjugate itself can be immobilized on a support, e.g., via non-covalent absorption of the carrier protein to a support surface. Such immobilized conjugates can find use in diagnostic applications.
  • glycan groups can be linked to a moiety of interest (e.g., as described herein) to provide an anti-GMl antibody-binding compound or conjugate capable of multivalent binding to the antibody.
  • the anti-GMl antibody-binding compound or conjugate is of formula (I) comprising one or more glycans linked to a moiety of interest: or a pharmaceutically acceptable salt thereof, wherein:
  • R 1 is a sialic acid group or an optionally substituted carboxymethyl group
  • Z 1 is -0-, -S-, -NR 2 - or -C(R 2 )2-, wherein each R 2 is independently selected from H, (C1-C4)- alkyl, (Ci-C 4 )-alkoxy, -CH 2 C 6 H 5 , -CH 2 CH 2 C6H 5 , -OCH 2 C 6 H 5 , and -OCH 2 CH 2 C 6 H 5 ;
  • Ar is optionally substituted aryl or optionally substituted heteroaryl (e.g., as described herein);
  • L 1 is a linker (e.g., as described herein); m is one or more; and
  • Y is a moiety of interest (e.g., as described herein).
  • m is 2 or more, such as 3 or more, 4 or more, 5 or more, 10 or more,
  • Y is a polymer (e.g., as described herein), and m depends on the length of the polymer and the % loading (i.e., loading based on a molar ratio). In some embodiments, m is 2 to 1000, such as 20 to 1000, 50 to 500, 50 to 400, 60 to 300, 60 to 200, 80 to 150 or 80 to 120.
  • Y is a support (e.g., as described herein), and m depends on the mass loading of glycan groups on the support.
  • the conjugate material can be characterized in terms of a loading in terms of mass ratio of glycan groups to the underlying support material.
  • the loading can be characterized in terms of the density of functional attachment points on the support.
  • the anti-GMl antibody-binding polymeric compound of formula (I) is of formula (II): wherein: q is 0 to 4; and each R 11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
  • the glycan group is as defined in any one of the embodiments of the glycan groups of formula (XI)-(XII) described here.
  • the compound is selected from formula (Ila)-(IIe):
  • -L 1 - is -Z 11 -(Ci-C 6 )-alkyl-Z 12 -(Ci-C 6 )-alkyl-S-CH 2 CO-;
  • -L 1 - comprises -(Ci-C6)-alkyl-amido-(Ci-C6)-alkyl-S-CH2CO-, and connects via an amide bond to an amine group of Y.
  • -L 1 - comprises - (CH2)2NHCO(CH2)3-S-CH2CO-, and connects via an amide bond to an amine group of Y.
  • the moiety of interest Y is a polymer.
  • Y is an amino acid polymer (e.g., polylysine). In some embodiments of formula (I)-(IIe), Y is an amino acid polymer backbone having a mean length of 10 to 5000 amino acid residues, such as 20 to 2000, 30 to 1000, or 50 to 800, 50 to 500, 100 to 500, 200 to 500, or 300 to 400 amino acid residues.
  • Y is an amino acid polymer backbone having a mean length of 360-440 amino acid residues, such as 380-420 amino acid residues, or 390-410 amino acid residues, e.g., about 400 amino acid residues.
  • the polymer is an alpha-amino acid polymer, and the polymer is composed of amino acid resides selected from lysine, ornithine, glutamic acid, aspartic acid, and serine.
  • the polymer is poly-lysine. In some embodiments of formula (I)-(IIe), the polymer is poly- -lysine. In some embodiments of formula (I)-(IIe), the polymer is poly-D-lysine. In some embodiments of formula (I)-(IIe), the polymer is a co-polymer of lysine residues and one or more additional amino acid residues (e.g., as described herein). In some embodiments of formula (I)-(IIe), the polymer is poly-aspartic acid or poly-glutamic acid.
  • the polymer is an alpha-amino acid polymer, and the percentage of loading of the glycan group onto the polymer backbone is 15 to 50%, such as 20 to 40%, or 25 to 40%. In some embodiments of formula (I)-(IIe), the loading of the glycan group onto the polymer backbone is 25 to 30%. In some embodiments of formula (I)-(IIe), the loading of the glycan group onto the polymer backbone is 30 to 40%, such as 30 to 35%, or 36 to 40%. In some embodiments of formula (I)-(IIe), the loading of the glycan group onto the polymer backbone is about 36%.
  • loading of the glycan group onto the polymer backbone is about 29%.
  • the polymer backbone is poly-lysine, such as poly- -lysine or poly-D-lysine.
  • the polymer is poly-lysine and further comprises spacer moieties attached to the amine side chain groups of the lysine residues to provide for linking to the glycan groups (e.g., of formula (V)).
  • spacer groups can be used to provide for linkage between alternative amino acid residues (such as aspartic acid or glutamic acid residues), and glycan-linker compounds of formula (V).
  • alternative amino acid residues such as aspartic acid or glutamic acid residues
  • glycan-linker compounds of formula (V) is conjugated directly to an amino acid side chain group with an intermediate spacer.
  • the polymer is poly-lysine polymer where a fraction of the lysine side chains are loaded with glycan groups (e.g., as described above) and substantially all of the remaining lysine side chains in the poly-lysine polymer are capped with a water solubilizing substituent.
  • the water solubilizing substituent attached to the lysine side chains is 2,3-dihydroxypropylthioacetyl-.
  • multimeric anti-GMl antibody-binding compounds or conjugates where multiple (e.g., two, three or more) glycan-containing compounds or conjugates are linked together, e.g., via linkage to their moieties of interest (Y).
  • Multimerization can be achieved via use of a branching moiety having two, three or more linked functional groups that provide points of attachment to which a suitable functional group of the moiety of interest (Y) can be conjugated.
  • Any suitable chemoselective ligation group chemistry e.g., as described herein can be used to connect a branching moiety to each monomeric anti-GMl antibody-binding compounds or conjugate.
  • the moiety of interest Y is a polymer.
  • a multimeric anti-GMl antibody-binding compound is of formula (IV): or a pharmaceutically acceptable salt thereof, wherein:
  • M 1 , M 2 and M 3 are each independently monomeric units that together provide a polymer backbone (P) (e.g., as described herein) having a mean length of 10-5000 amino acid residues, x is 10 to 50 mol% of M 1 units in the polymer backbone (P); y is 0 to 90 mol% of M 2 units in the polymer backbone (P);
  • P polymer backbone
  • Y 2 is an optional terminal group
  • L 1 is a linker that connects a glycan (G) to the M 1 monomeric unit;
  • WSG is a water solubilizing group linked to the M 2 monomeric unit
  • L 2 is an optional linker connecting the polymer backbone (P) to B;
  • B is a branching moiety that covalently links to “n” polymer backbones, wherein n is 2 to 6; and each G is independently a glycan group that mimics GM1.
  • the G groups linked to the polymer backbone (P) include at least one glycan group according to any one of the embodiments of formula (XI) -(XII) as described herein.
  • each G is independently of formula (XI) or (XIV) or wherein:
  • R 1 is a sialic acid group or an optionally substituted carboxymethyl group.
  • Z 1 and Z 3 are selected from -0-, -S-, -NR 2 - and -C(R 2 )2-, wherein each R 2 is independently selected from H, (Ci-C -alkyl, (Ci-C- -alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and - OCH2CH2C6H5; and
  • Ar is selected from optionally substituted aryl, and optionally substituted heteroaryl.
  • the G groups linked to the polymer backbone (P) include at least one glycan group of formula (XIV).
  • R 1 is a sialic acid group, e.g., of formula (XHIa) as described herein.
  • R 1 is optionally substituted carboxymethyl group, e.g., of formula (XHIb) as described herein.
  • M 1 , M 2 and M 3 are each independently monomeric units of a polymer selected from amino acid polymers, polyacrylate polymers or copolymers, poly(meth)acrylate polymers or copolymers, N-vinyl-2-pyrrolidone-vinylalcohol copolymers, chitosan polymers, and polyphosphazene polymers.
  • M 1 , M 2 and M 3 are each independently monomeric units that together provide a polymer backbone (P) having a mean length of 20 to 2000 repeat units, such as 30 to 1000, 50 to 800, 50 to 300, 50 to 200, or 100 to 200 repeat units.
  • the M 1 monomer units can include a sidechain functional group capable of conjugation to a linked glycan group (e.g., as described herein).
  • the M2 monomer units can include a sidechain functional group capable of conjugation to a WSG (e.g., as described herein).
  • the M 2 monomer units selected for incorporation into the polymer backbone already have a hydrophilic or charged WSG sidechain group as part of the repeat unit.
  • Any convenient M 3 monomer units can be selected to impart a desirable property upon the polymer backbone.
  • the M 1 , M 2 and M 3 monomer units are all of the same monomer class and monomer chemistry.
  • the M 1 , M 2 and/or M 3 monomer units are assembled via a random polymerization by combining desired ratios of the monomer units for polymerization. In some embodiments, the M 1 , M 2 and/or M 3 monomer units are assembled via a block copolymer polymerization method. In some embodiments, the M 1 , M 2 and/or M 3 monomer units are assembled in a stepwise fashion and have a defined length and sequence. [0142] In some embodiments of formula (IV), the multimeric compound includes ‘m’ polymer backbones (P) that together have a total number of repeat units of 150 to 1000, such as 150 to 8000, 150 to 600, or 300 to 600 repeat units.
  • P polymer backbones
  • M 1 , M 2 and M 3 are each independently amino acid residues that together provide an amino acid polymer backbone (P) having a mean length of 20 to 2000 amino acid residues, such as 30 to 1000, 50 to 800, 50 to 300, 50 to 200, or 100 to 200 amino acid residues.
  • the multimeric compound includes ‘m’ amino acid polymer backbones (P) that together have a total number of amino acid residues of 150 to 1000, such as 150 to 8000, 150 to 600, or 300 to 600 amino acid residues.
  • x is 15 to 50 mol% of M 1 units in the polymer backbone (P), such as 20 to 40 mol% or 25 to 40 mol%, or 30 to 40 mol%.
  • x + y is 1, such that M 3 is absent.
  • M 3 is present, and in some cases, present at 1 to 25 mol%, such as 1 to 20 mol%, or 1 to 10 mol%.
  • y is > 0 mol%. In some embodiments of formula (IV), y is at least 10 mol%, such as at least 20 mol%, at least 40 mol%, or at least 50 mol%.
  • y is 50 to 75 mol%, such as 60 to 75 mol%.
  • Y 2 is an optional terminal group.
  • a terminal group can be any suitable group or moiety, such as H-, a terminal group of a monomer, a capping group, a protecting group, a linked glycan, a linked moiety of interest, or a residue of polymer synthesis, such as a polymerization initiator.
  • a linker e.g., L2
  • L2 can be connected to a terminal of the polymer, e.g., directly to a terminal monomer unit, or via an optional terminal group, such as a residue of a polymerization initiator (e.g., as described herein).
  • the polymer P is a linear amino acid polymer, and Y 2 is a terminal group located at the N-terminal amino acid residue. In some embodiments of formula (IV), the polymer P is a linear amino acid polymer, and Y 2 is a terminal group located at the C- terminal amino acid residue.
  • the polymer P is an amino acid polymer where each M 1 is an amino acid that includes a sidechain group capable of conjugation (e.g., via chemoselective ligation) to a -L'-G is a glycan group of formula (XI)-(XII) (e.g., as described herein).
  • each M 1 is selected from lysine, ornithine, aspartic acid, and glutamic acid, where the glycan group can be attached via amide bond formation.
  • each M 1 is an amino acid residue having a sidechain group that has been modified with an intermediate linking group that install a desirable sidechain chemosselective ligation group compatible with a complementary group of the glycan-linker precursor.
  • a lysine monomer unit having a sidechain modified with a thiol-reactive group e.g., a haloacetamide group (- COCH2CI).
  • M 2 can be any convenient amino acid unit that is capable of conjugation to a water solubilizing group.
  • each M 2 is selected from lysine, ornithine, aspartic acid, and glutamic acid, where the water solubilizing group can be attached via amide bond formation.
  • each M 2 is an amino acid residue having a sidechain group that has been modified with an intermediate linking group that install a desirable sidechain chemoselective ligation group compatible with a complementary group of the WSG precursor.
  • a lysine monomer unit having a sidechain modified with a thiol-reactive group e.g., a haloacetamide group (-COCH2CI).
  • the M 3 repeat unit can be any convenient amino acid.
  • the M3 can be present at a sufficient level (mol %) that together with the M 1 and M 2 repeat units provide a compound having desirable glycan loading and/or physical properties.
  • M 3 is an amino acid residue selected from glycine, alanine, or serine.
  • the polymer P is an amino acid polymer where each M 1 is lysine residue, and each -L'-G is a glycan group of formula (XI)-(XII) (e.g., as described herein).
  • the polymer P is an amino acid polymer where each M 2 is lysine residue linked to a water solubilizing group via a spacer (e.g., haloacetyl spacer conjugated with a thiol containing water soluble group (e.g., thioglycerol).
  • M 3 is absent and M 1 and M 2 are lysine residues.
  • multimeric anti-GMl antibody-binding compound of formula (IV) may be represented by an alternative formula, e.g., a formula as follows that depicts the several glycan groups linked to the polymer backbone, which is itself multimerized.
  • the anti-GMl antibody-binding multimeric compound is of formula (III): or a pharmaceutically acceptable salt thereof, wherein:
  • R 1 is a sialic acid group or an optionally substituted carboxymethyl group.
  • Z 1 is -0-, -S-, -NR 2 - or -C(R 2 ) 2 -, wherein each R 2 is independently selected from H, (C1-C4)- alkyl, (Ci-C 4 )-alkoxy, -CH 2 C 6 H 5 , -CH 2 CH 2 C 6 H 5 , -OCH 2 C 6 H 5 , and -OCH 2 CH 2 C 6 H 5 ;
  • Ar is optionally substituted aryl or optionally substituted heteroaryl (e.g., monocyclic aryl or heteroaryl, or bicyclic aryl or heteroaryl);
  • L 1 is a linker; m is at least 2;
  • P is a polymer
  • L 2 is an optional linker; n is 2 to 6; and B is a branching moiety.
  • n is 2 whereby the compound is dimeric. In some embodiments, the compound is homo-dimeric. In some embodiments, the compound is hetero- dimeric.
  • n is 3 whereby the compound is trimeric.
  • Higher valency multimers can also be provided, e.g., by configuration of multiple branching moieties that produce a dendrimer.
  • the branching moiety B can be a distinct and different moiety from the polymer P, e.g., B is a group or molecule that is different from any repeat unit of the polymer P, and can be attached to the polymer P via an orthogonal chemistry as compared to the chemoselective ligation chemistry used to achieve attachment of the gly can-linker compounds to the polymer backbone.
  • B is aryl or heteroaryl group substituted at two or more positions with L 2 linkers, and optionally further substituted.
  • B is derived from an aryl or heteroaryl group substituted at two or more positions with carboxylic acid groups, or derivatives thereof, to which optional linkers (L 2 ) can be attached, and optionally further substituted.
  • B is optionally substituted phenyl substituted at two or more positions via linking moieties to L 2 linkers, where the L 2 linkers can include chemoselective ligation groups for attachment to P polymers.
  • B is a trisubstituted aryl or heteroaryl group, and L 2 is -CONH-(Ci-Cg)-alkyl-NH-.
  • B is a trisubstituted phenyl group, and L 2 is -CONH-CH2CH2-NH-.
  • P is an amino acid polymer (e.g., poly-lysine) and L 2 connects to the C-terminal of the polymer via an amide bond.
  • each carbonyl group links to an L 2 linker of formula (IV)-(III), e.g., via an amide linkage.
  • each P is an amino acid polymer backbone (e.g., as described herein), and each L 2 links the C-terminal residue of the amino acid polymer backbone to a carbonyl or carboxyl group of B via amide bonds.
  • the amino acid polymer backbone is linear.
  • each P is an amino acid polymer backbone
  • each L 2 links the N-terminal residue of the a-amino acid polymer backbone to B.
  • the amino acid polymer backbone is linear.
  • Any suitable linkers e.g., as described herein can be incorporated into the compounds of formula (III) to link the branching moiety B with the two, three or more polymers P.
  • each L 2 is a linear linker comprising one or more linking moieties independently selected from -Ci- 6 -alkylene-, -NHCO-Ci- 6 -alkylene-, -CONH-C I-6 - alkylene-, -0(CH 2 ) p- , -(OCH 2 CH 2 ) p- , -NHCO-, -CONH-, -NHS0 2- , -S0 2 NH-, -CO-, -S0 2-
  • each L 2 is -NH-(CH 2 ) P -NH-, wherein p is 2 to 6. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4.
  • Ar is wherein: q is 0 to 4; and each R 11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
  • the compound includes a multitude of glycan- linker groups of formula (XI)-(XII), as defined in any one of the embodiments described herein.
  • exemplary polymeric compounds of formula (I)-(IIe), are described in the experimental section. 5.1.8.
  • compositions comprising a compound (e.g., as described herein).
  • the pharmaceutical composition includes a polymeric compound of formula (I)-(IV), e.g., as described by any of the embodiments set forth herein.
  • compositions for parenteral administration such as subcutaneous, intravenous, intrahepatic or intramuscular administration, to warm-blooded animals, especially humans, are considered.
  • the compositions comprise the active ingredient(s) alone or, preferably, together with a pharmaceutically acceptable carrier.
  • the dosage of the active ingredient(s) depends upon the age, weight, and individual condition of the patient, the individual pharmacokinetic data, and the mode of administration.
  • compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, viscosity-increasing agents, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.
  • Suitable carriers for enteral administration are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example com, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate.
  • Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or poly
  • Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinyl-pyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl-methylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient(s).
  • suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinyl-pyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl-
  • compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the hard capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as com starch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers.
  • the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.
  • suitable liquid excipients such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.
  • compositions according to this disclosure may contain separate tablets, granules or other forms of orally acceptable formulation of the active ingredients, or may contain a mixture of active ingredients in one suitable pharmaceutical dosage form, as described above.
  • the separate orally acceptable formulations or the mixture in one suitable pharmaceutical dosage form may be slow release and controlled release pharmaceutical compositions.
  • the pharmaceutical compositions can comprise from approximately 0.1% to approximately 95% active ingredient or mixture of active ingredients. In some embodiments, the composition is in a single-dose administration form.
  • This disclosure also relates to the mentioned pharmaceutical compositions as medicaments in the treatment of neurological diseases associated with anti-glycan antibodies, particularly immune- mediated neuropathies.
  • Another aspect of this disclosure relates to a method of inhibiting or specifically binding an anti-GMl antibody in a sample.
  • the method includes contacting a sample comprising the anti-GMl antibody with an effective amount of a compound or conjugate of this disclosure (e.g., of formula (I)-(IV) as described herein).
  • the compound or conjugate is a polymeric compound of formula (I) where Y is a polymer, such as an amino acid polymer (e.g., as described herein).
  • the compound or conjugate is a conjugate of formula (I) where Y is a support.
  • the sample is a biological sample.
  • the biological sample can be obtained from a subject and can be any suitable body fluid sample.
  • Body fluids that are useful for binding of anti-GMl antibodies include, but are not limited to, serum, plasma, whole blood, cerebrospinal fluid, and extracts from solid tissue.
  • the biological sample is serum or plasma.
  • the anti-GMl antibody mediated neuropathy is Guillain-Barre-Syndrome (GBS), including variants of GBS such as acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) or a pharyngeal-cervical-brachial variant of GBS, or the chronic multifocal motor neuropathy (MMN).
  • GBS Guillain-Barre-Syndrome
  • AMAN acute motor axonal neuropathy
  • AMSAN acute motor-sensory axonal neuropathy
  • AIDP acute inflammatory demyelinating polyneuropathy
  • MNN chronic multifocal motor neuropathy
  • the present invention relates furthermore to a method of treatment of neurological diseases associated with anti-glycan antibodies, particularly immune-mediated neuropathies, which comprises administering a composition according to this disclosure in a quantity effective against said disease, to a warm-blooded animal requiring such treatment.
  • the pharmaceutical compositions can be administered prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment.
  • the daily, weekly or monthly dose administered is from approximately 0.01 g to approximately 5 g, preferably from approximately 0.1 g to approximately 1.5 g, of the active ingredients in a composition of the present invention.
  • This disclosure includes methods of treatment of neurological diseases associated with anti- glycan antibodies, particularly immune-mediated neuropathies.
  • the methods can include extracorporeal separation of target anti-GMl autoantibodies from a biological sample, e.g. a biological fluid such as peripheral blood (or other biological fluids such as whole blood, arterial blood, cerebrospinal fluid, peritoneal or pleural fluid, etc.) of a warm-blooded animal (for example a human patient) requiring such treatment.
  • a biological fluid such as peripheral blood (or other biological fluids such as whole blood, arterial blood, cerebrospinal fluid, peritoneal or pleural fluid, etc.) of a warm-blooded animal (for example a human patient) requiring such treatment.
  • the extracorporeal separation of target anti-GMl autoantibodies from a biological sample of a patient can be achieved by contacting the sample with an effective amount of a compound of this disclosure that is immobilized on a support in a format suitable for achieving separation.
  • the support is beads.
  • the beads are packed in a column or cartridge, and the biological sample is flowed through the column or cartridge to remove the target anti-GMl autoantibodies from the biological sample.
  • the beads are magnetic and subsequent application of a strong magnet will pull down the beads along with the target anti-GMl autoantibodies, allowing those antibodies to be removed from the biological sample.
  • the biological sample e.g., peripheral blood
  • the beads are biocompatible and suitable for use in an ex vivo application.
  • the immobilized compositions e.g., beads
  • the immobilized compositions can be utilized prophylactically or therapeutically.
  • Another aspect of this disclosure relates to a method of diagnosis of neurological diseases, particularly immune-mediated neuropathies, wherein the level of antibodies (e.g. IgM/IgG) against glycans of the nervous system, particularly GM1 ganglioside, is determined in a biological sample.
  • a high level of anti-GMl autoantibodies in the sample is indicative of the development and the severity of a particular anti-GMl antibody mediated neuropathy.
  • the anti-GMl antibody mediated neuropathy is Guillain-Barre-Syndrome (GBS), including variants of GBS such as acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) or a pharyngeal-cervical-brachial variant of GBS, or the chronic multifocal motor neuropathy (MMN).
  • GBS Guillain-Barre-Syndrome
  • AMAN acute motor axonal neuropathy
  • AMSAN acute motor-sensory axonal neuropathy
  • AIDP acute inflammatory demyelinating polyneuropathy
  • MNN chronic multifocal motor neuropathy
  • the biological sample is obtained from the patient and can be any suitable body fluid sample.
  • Body fluids that are useful for determination of antibodies against GM1 glycoepitopes include, but are not limited to, serum, plasma, whole blood, cerebrospinal fluid, and extracts from solid tissue.
  • the biological sample is serum.
  • Any suitable method may be used for the determination of the level of anti-GMl autoantibodies in the biological sample. Methods considered are, e.g., ELISA, RIA, EIA, Flow Cytometry, electrochemiluminescence system or microarray analysis.
  • the method used for the determination of anti-GMl autoantibodies in the biological sample involves capture of the autoantibodies with beads loaded with compounds of this disclosure.
  • the beads utilized in the diagnostic assay include gly can-linker compounds of formula (V).
  • the beads can be adapted for use in a variety of assays to qualitatively or quantitatively assess the presence or level of anti-GMl autoantibodies in the sample.
  • anti-GMl autoantibodies bound to the glycan-linked beads can be detected using a secondary antibody labelled with a dye.
  • an ELISA assay is performed in the bead format to detect bead bound anti-GMl autoantibodies.
  • a flow cytometry assay is performed in the bead format to detect bead bound anti-GMl autoantibodies. Detection can be achieved using a variety of methods, including flow cytometry, or a bead based sandwich assay with fluorescence microscopy.
  • the method used for the determination of anti-GMl autoantibodies in human body fluids is an ELISA.
  • microtiter plates are coated with glycan-linker compounds of formula (V) or preferably conjugates of this disclosure (e.g., peptidic compounds, or carrier molecule conjugates, or the like) comprising such compounds of formula (V) as substituents.
  • the plates are then blocked and the biological sample or a standard solution is loaded.
  • an anti-IgM/IgG antibody is applied, e.g. an anti-IgM or anti-IgG antibody directly conjugated with a suitable label, e.g. with an enzyme for chromogenic detection.
  • a polyclonal rabbit (or mouse) anti-IgM / anti-IgG antibody is added.
  • a second antibody detecting the particular type of the anti-IgM / anti-IgG antibody, e.g. an anti-rabbit (or anti mouse) antibody, conjugated with a suitable label, e.g. the enzyme for chromogenic detection as above, is then added.
  • the plate is developed with a substrate for the label in order to detect and quantify the label, being a measure for the presence and amount of anti-GMl autoantibodies.
  • the label is an enzyme for chromogenic detection
  • the substrate is a colour-generating substrate of the conjugated enzyme. The colour reaction is then detected in a microplate reader and compared to standards.
  • Suitable labels are chromogenic labels, i.e. enzymes which can be used to convert a substrate to a detectable colored or fluorescent compound, spectroscopic labels, e.g. fluorescent labels or labels presenting a visible color, affinity labels which may be developed by a further compound specific for the label and allowing easy detection and quantification, or any other label used in standard ELISA.
  • chromogenic labels i.e. enzymes which can be used to convert a substrate to a detectable colored or fluorescent compound
  • spectroscopic labels e.g. fluorescent labels or labels presenting a visible color
  • affinity labels which may be developed by a further compound specific for the label and allowing easy detection and quantification, or any other label used in standard ELISA.
  • target antibodies are radioimmunoassay or competitive immunoassay and chemiluminescence or electrochemiluminescence detection on automated commercial analytical robots. Microparticle enhanced fluorescence, fluorescence polarized methodologies, or mass spectrometry may also be used. Detection devices, e.g. microarrays, are useful components as readout systems for target antibodies.
  • the kits can further contain anti-IgM / anti-IgG antibodies (or anti-IgM/IgG antibody fragments) carrying a suitable label, or anti-IgM / anti-IgG antibodies and second antibodies carrying such a suitable label, and/or reagents or equipment to detect the label, e.g. reagents reacting with enzymes used as labels and indicating the presence of such a label by a color formation or fluorescence, standard equipment, such as microtiter plates, pipettes and the like, standard solutions and wash solutions.
  • the ELISA can be also designed in a way that patient blood or serum samples are used for the coating of microtiter plates with the subsequent detection of anti-GMl antibodies with labelled compounds of formula (V) (e.g., a compound linked to a detectable label) or conjugates or polymeric compounds comprising such compounds as substituents, and a detectable label.
  • the label is either directly detectable or indirectly detectable, e.g., via an antibody or other binding protein.
  • the detectable label is a fluorophore or chromophore dye.
  • glyco coepitope refers to the carbohydrate moiety or glycan group that is recognized by an anti-GMl antibody.
  • reducing end refers to the monosaccharide of the glycoepitope with a free anomeric carbon that is not involved in a glycosidic bond, wherein said free anomeric carbon bears a hemiacetal group.
  • (Ci-C n )-alkyl refers to straight or branched carbon chain of 1 to n carbon atoms.
  • the term “(Ci-C4)-alkyl” includes butyl, such as /7-butyl sec-butyl, Ao-butyl, tert- butyl, propyl, such as «-propyl or iso- propyl, ethyl or methyl.
  • the term “(Ci-C 4 )-alkyl” refers to methyl or ethyl, «-propyl or 750-propyl.
  • the term “(Ci-C 4 )-alkyl” refers to methyl.
  • CVCValkyl refers to straight or branched chain of 1 to 6 carbon atoms.
  • (Ci-C n )-alkylene“ refers to a straight or branched bivalent alkyl chain of 1 to n carbon atoms, and includes, for example, -CH 2 -, -CH 2 -CH 2 -, -CH(CH3)-, -CH 2 -CH 2 -CH 2 -, -CH(CH3)- CH 2 -, or -CH(CH 2 CH 3 )-, etc.
  • (Ci-C n )-alkoxy refers to an alkoxy with a straight or branched chain of 1 to n carbon atoms.
  • the term “Ci-C 4 -alkoxy”, as used herein refers to methoxy, ethoxy, propoxy.
  • the term “Ci-C 4 -alkoxy”, as used herein, refers to methoxy.
  • Double bonds in principle can have E- or Z-configuration.
  • the compounds of this invention may therefore exist as isomeric mixtures or single isomers. If not specified both isomeric forms are intended.
  • Any asymmetric carbon atoms may be present in the ( R )-, (.V)- or (R, ⁇ S) -configuration, preferably in the ( R )- or ( ⁇ -configuration.
  • the compounds may thus be present as mixtures of isomers or as pure isomers, preferably as enantiomer-pure diastereomers.
  • alkynyl refers to is a straight or branched carbon chain or cyclic group comprising one or more, preferably one triple bond. Preferred are Ci-C 4 -alkynyl, such as propargyl or acetylenyl.
  • An exemplary cyclic alkynyl group is a cyclooctyne, or substituted cyclooctyne.
  • aryl refers to a mono- or bicyclic system of aromatic rings with 5 to 12 carbon atoms optionally carrying substituents, such as phenyl, 1 -naphthyl or 2-naphthyl, or also a partially saturated bicyclic fused ring comprising a phenyl group, such as indanyl, indolinyl, dihydro- or tetrahydronaphthyl, all optionally substituted.
  • substituents such as phenyl, 1 -naphthyl or 2-naphthyl
  • aryl group such as indanyl, indolinyl, dihydro- or tetrahydronaphthyl, all optionally substituted.
  • aryl is phenyl, indanyl, indolinyl or tetrahydronaphthyl, in particular phenyl.
  • heteroaryl refers to an aromatic mono- or bicyclic ring system containing at least one heteroatom, and preferably up to three heteroatoms selected from nitrogen, oxygen and sulfur as ring members. Heteroaryl rings do not contain adjacent oxygen atoms, adjacent sulfur atoms, or adjacent oxygen and sulfur atoms within the ring.
  • Monocyclic heteroaryl preferably refers to 5 or 6 membered heteroaryl groups and bicyclic heteroaryl preferably refers to 9 or 10 membered fused-ring heteroaryl groups.
  • heteroaryl examples include pyrrolyl, thienyl, fiiryl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, and benzo or pyridazo fused derivatives of such monocyclic heteroaryl groups, such as indolyl, benzimidazolyl, benzofuryl, quinolinyl, isoquinolinyl, quinazolinyl, pyrrolopyridine, imidazopyridine, or purinyl, all optionally substituted.
  • heteroaryl refers to a 5- or 6-membered aromatic monocyclic ring system containing at least one heteroatom, and preferably up to three heteroatoms selected from nitrogen, oxygen and sulfur as ring members.
  • heteroaryl is pyridyl, pyrimdinyl, pyrazinyl, pyridazinyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, oxadiazolyl, triazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyrrolyl, indolyl, pyrrolopyridine or imidazopyridine; in particular pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, imidazolyl, thiazolyl, oxadiazolyl, triazolyl, indolyl, pyrrolopyridine or imidazopyridine
  • optionally substituted aryl refers to aryl substituted by up to five substituents, preferably up to two substituents.
  • substituents are preferably and independently selected from Ci-C t -alkyl, Ci-C t -alkoxy, amino-Ci-Cz t -alkyl, acylamino-Ci-C t -alkyl, aryl-Ci-C t -alkyl hydroxy, carboxy, cyano, C1-C4- alkoxycarbonyl, aminocarbonyl, hydroxylaminocarbonyl, tetrazolyl, hydroxysulfonyl, aminosulfonyl, hydroxy, halo, or nitro, in particular Ci-C t -alkyl, Ci-C t -alkoxy, amino-Ci-C t -alkyl, acyla
  • optionally substituted heteroaryl refers to heteroaryl substituted by up to three substituents, preferably up to two substituents.
  • substituents are preferably and independently selected from Ci-C t -alkyl, Ci-C t -alkoxy, halo-Ci-C t -alkyl, hydroxy, Ci- C t -alkoxycarbonyl, aminocarbonyl, hydroxylaminocarbonyl, tetrazolyl, aminosulfonyl, halo, aryl-Ci- C t -alkyl, or nitro.
  • Cycloalkyl has preferably 3 to 7 ring carbon atoms, and may be unsubstituted or substituted, e.g. by Ci-C t -alkyl or Ci-C t -alkoxy. Cycloalkyl is, for example and preferably, cyclohexyl, cyclopentyl, methylcyclopentyl, or cyclopropyl, in particular cyclopropyl.
  • Acyl designates, for example, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, aryl- C1-C4- alkylcarbonyl, or heteroarylcarbonyl.
  • Ci-C4-acyl is preferably lower alkylcarbonyl, in particular propionyl or acetyl.
  • Ac stands for acetyl.
  • Hydroxyalkyl is especially hydroxy-(Ci-C4)-alkyl, preferably hydroxymethyl, 2-hydroxyethyl or 2-hydroxy-2 -propyl.
  • Haloalkyl is preferably fluoroalkyl, especially trifluoromethyl, 3,3,3-trifluoroethyl or pentafluoroethyl.
  • Halogen is fluorine, chlorine, bromine, or iodine.
  • Arylalkyl includes aryl and alkyl groups, e.g. benzyl, 1-phenethyl or 2-phenethyl.
  • Heteroarylalkyl includes heteroaryl and alkyl as defined hereinbefore, and is e.g. 2-, 3- or 4- pyridylmethyl, 1- or 2-pyrrolylmethyl, 1-pyrazolylmethyl, 1-imidazolylmethyl, 2-(l-imidazolyl)ethyl or 3-(l-imidazolyl)propyl.
  • substituted amino the substituents are preferably those mentioned as substituents hereinbefore.
  • substituted amino is alkylamino, dialkylamino, optionally substituted arylamino, optionally substituted arylalkylamino, lower alkylcarbonylamino, benzoylamino, pyridylcarbonylamino, lower alkoxycarbonylamino or optionally substituted aminocarbonylamino.
  • Particular salts considered are those replacing the hydrogen atoms of a carboxylic acid function. Suitable cations are, e.g., sodium, potassium, calcium, magnesium or ammonium cations.
  • any reference to the free compounds herein is to be understood as referring also to the corresponding salts, and vice versa, as appropriate and expedient.
  • NMR spectra were recorded on a Bruker Avance DMX-500 (500.1 MHz) spectrometer. Assignment of 'H and 13 C NMR spectra was achieved using 2D methods (COSY, HSQC, HMBC). Chemical shifts are expressed in ppm using residual CHCh, CHD2OD or HDO as references. Electron spray ionization mass spectra (ESI-MS) were obtained on a Waters micromass ZQ. The LC/HRMS analyses were carried out using an Agilent 1100 LC equipped with a photodiode array detector and a Micromass QTOF I equipped with a 4 GHz digital-time converter.
  • Reactions were monitored by TLC using glass plates coated with silica gel 60 F254 (Merck) and visualized by using UV light and/or by charring with a molybdate solution (a 0.02 M solution of ammonium cerium sulfate dihydrate and ammonium molybdate tetrahydrate in aqueous 10% H2SO4).
  • MPLC separations were carried out on a CombiFlash Companion or Rf from Teledyne Isco equipped with RediSep normal -phase or RP-18 reversed-phase flash columns.
  • LC-MS separations were done on a Waters system equipped with sample manager 2767, pump 2525, PDA 2525 and micromass ZQ.
  • Scheme 2 outlines the representative synthesis of the propionic acid analogues and the triflate building blocks (20 and 22), which involves the conversion of amino- to hydroxyl-group under Sandmeyer Reaction condition (NaNCE/ELSCE), benzylation of the carboxylic acid, and triflation.
  • Compound 2 shares most of the structural features as natural GM1 epitope (1), except a tyramine moiety instead of amino-propyl glucoside (Part-I).
  • Part-I tyramine moiety instead of amino-propyl glucoside
  • Peracetylated sialyl donor (24) is known to have lower reactivity than common pyranosyl donors, due to the electron-withdrawing carbonyl group presenting at the anomeric carbon, therefore primarily reacts with the more reactive 3 -OH on the galactosyl acceptor (23a-b).
  • GalN donor (13) and sialosyl acceptors (25a-b) proceeded in 4-5 hours and gave fully-protected nat-GMl and GM1 mimetic (26a-b) in good yields.
  • Acetyl- and benzoyl- protection of hydroxyls and NTroc were removed at once under basic condition at elevated temperature, and the acetamide was introduced to the 2-NH2 of GalN fragment with Ac 2 0/MeOH/triethylamine.
  • Both hydrogenation conditions such as in-situ hydrogen generation with ammonium formate/palladium black (®natGMl 1), and palladium hydroxide/charcoal catalyzed hydrogenation (®GM1 mimetic 2), successfully removed the benzyl and Cbz protecting groups to yield the target oligosaccharides (1-2).
  • the monomeric GM1 analogues (1-9) were firstly reacted with thiobutyrolactone and triethylamine to attach a mercapto-butanamide linker ( >40-48) .
  • the commercial linear or branched polylysines (linear 49-53 and branched 83-85, Scheme 7 and 8) were chloroacetylated, giving the activated polylysines - structurally represented as 54-58 for the linear and 86-88 for the branched polymers.
  • Compound 16 Compound 14 (500 mg, 0.79 mmol), 15 (285 mg, 0.55 mmol) and freshly activated 4 ⁇ molecular sieves were mixed in DCM/petrol ether (10 mL, 2: 1). The mixture was cooled to -10°C, and then added TMSOTf (17 mg, 0.08 mmol) drop wise. The reaction mixture was stirred at -10°C for 10 min then quenched with triethylamine. The reaction mixture was fdtered and concentrated, and the residue was purified on silica gel MPLC with 30-40% EtOAc/petrol ether to give the product 16 as white solid (710 mg, yield: 40%). TLC condition: Petrol ether / EtOAc (3:2),
  • Compound 17 Compound 16 (710 mg, 0.72 mmol) was dissolved in MeOH (20mL) and p- toluenesulfonic acid monohydrate (204 mg, 1.07 mmol) with water (250pmL) was added at RT. The solution was stirred overnight and then quenched with triethylamine (0.5 mL). The reaction mixture was vacuum dried and the residue was treated with pyridine (10 mL). To the above mixture, AC2O (1.09 g, 10.7 mmol) and 4-DMAP (8.7 mg, 0.07 mmol) was added at 0°C.
  • the product was precipitated by drop-wise addition of the reaction mixture to vigorously stirring solvent mixture (ethanol/diethyl ether 1 : 1, 40 mL). The mixture was centrifuged at 1000 rpm, 4-10 °C, for 2 min. The solvent was decanted, and the residue was re-suspended in ethanol/ether (1:1, 30 mL). The centrifugation and re-suspension was repeated three times. The product precipitates were collected and dried under vacuum overnight to give the products 54-58, 86-88 as white solids (77-98%). 1 HNMR data were identical to the previous report. [Ref: G. Thoma et al., J. Am. Chem. Soc. 1999, 121: 5919-5929]
  • Polymer 59 natGMl epitope (l)-polymer 56 conjugate, monomer loading: 26%.
  • Polymer 60 GM1 mimetic (2)-polymer 56 conjugate, monomer loading: 30%.
  • Polymer 61 GM1 mimetic (3)-polymer 56 conjugate, monomer loading: 24%.
  • Polymer 62 GM1 mimetic (4)-polymer 56 conjugate, monomer loading: 25%.
  • Polymer 63 GM1 mimetic (5)-polymer 56 conjugate, monomer loading: 25%.
  • Polymer 64 GM1 mimetic (6)-polymer 56 conjugate, monomer loading: 26%.
  • Polymer 65 GM1 mimetic (7)-polymer 56 conjugate, monomer loading: 26%.
  • Polymer 66 GM1 mimetic (8)-polymer 56 conjugate, monomer loading: 35%.
  • Polymer 67 GM1 mimetic (9)-polymer 56 conjugate, monomer loading: 30%.
  • Polymer 68 GM1 mimetics (2/4, l:2)-polymer 56 conjugate, monomer loading: 30%.
  • Polymer 69 GM1 mimetic (2)-polymer 54 conjugate, monomer loading: 14%.
  • Polymer 70 GM1 mimetic (2)-polymer 54 conjugate, monomer loading: 30%.
  • Polymer 71 GM1 mimetic (2)-polymer 54 conjugate, monomer loading: 37%.
  • Polymer 72 GM1 mimetic (2)-polymer 55 conjugate, monomer loading: 14%.
  • Polymer 73 GM1 mimetic (2)-polymer 55 conjugate, monomer loading: 29%.
  • Polymer 74 GM1 mimetic (2)-polymer 55 conjugate, monomer loading: 37%.
  • Polymer 75 GM1 mimetic (2)-polymer 56 conjugate, monomer loading: 12%.
  • Polymer 76 GM1 mimetic (2)-polymer 56 conjugate, monomer loading: 37%.
  • Polymer 77 GM1 mimetic (2)-polymer 57 conjugate, monomer loading: 14%.
  • Polymer 78 GM1 mimetic (2)-polymer 57 conjugate, monomer loading: 28%.
  • Polymer 79 GM1 mimetic (2)-polymer 57 conjugate, monomer loading: 36%.
  • Polymer 80 GM1 mimetic (2)-polymer 58 conjugate, monomer loading: 12%.
  • Polymer 81 GM1 mimetic (2)-polymer 58 conjugate, monomer loading: 28%.
  • Polymer 82 GM1 mimetic (2)-polymer 58 conjugate, monomer loading: 36%.
  • Polymer 90 GM1 mimetic (2)-polymer 86 conjugate, monomer loading: 29%.
  • Polymer 91 GM1 mimetic (2)-polymer 86 conjugate, monomer loading: 37%.
  • Polymer 92 GM1 mimetic (2)-polymer 87 conjugate, monomer loading: 14%.
  • Polymer 93 GM1 mimetic (2)-polymer 87 conjugate, monomer loading: 29%.
  • Polymer 94 GM1 mimetic (2)-polymer 87 conjugate, monomer loading: 36%.
  • Polymer 96 GM1 mimetic (2)-polymer 88 conjugate, monomer loading: 29%.
  • Polymer 97 GM1 mimetic (2)-polymer 88 conjugate, monomer loading: 36%.
  • GM1 mimetic (2)-fimctionalized Sepharose beads 98 Epoxy-activated Sepharose 6B (0.5 g, 1.75 ml final volume, 70 mhio ⁇ active groups) was suspended in 100 ml distilled water for lh in 10x10 ml aliquots and filtered.
  • GM1 mimetic (2) (34.2 mg, 0.035 mmol, 0.50 eq) was dissolved in 1.75 ml carbonate buffer (0.1 M carbonate buffer + 0.15 M NaCl) and added to the Epoxy-activated Sepharose 6B in a closed vial. The mixture was shaken for 24 h at RT.
  • 2-aminoethanethiol (27 mg, 0.35 mmol, 5.00 eq, 1M in buffer) was added for capping and the mixture was shaken for another 16h at RT.
  • the mixture was filtered and washed 3 times with alternatively 0.1 M NaOAc pH 4 containing 0.5 M NaCl followed by 0.1 M Tris- HC1 pH 8 containing 0.5 M NaCl, and dried on the glass frit.
  • Sera from GBS, MMN and control neuropathy patients or healthy individuals were investigated. They were tested for anti-GMl IgG and IgM antibodies by ELISA. Serum anti-GMl antibody titers were determined by an ELISA assay from Biihlmann Laboratories (Schonenbuch, Switzerland). Sera were either obtained from University Medical Center Utrecht (Utrecht, Netherlands) or the immunology laboratory of the University Hospital Marseille (Marseille, Prance). Sera from healthy individuals (without neuropathy) or from control neuropathy patients both negative for anti-GMl reactivity served as control and were obtained from the blood bank in Basel (Blutspendetechnik SRK beider Basel, Basel, Switzerland). All participants signed an informed consent.
  • the blocking buffer was discarded without a washing step prior adding the 100 pL human serum diluted 1:50 in incubation buffer (2% BSA, 0.05% Tween20 in PBS) for two hours at 4- 8°C. After the incubation step, the plates were washed four times with 300 pL/well Wash Buffer and incubated for two hours at 4-8°C with 100 pL/we 11 of undiluted horseradish peroxidase-labeled anti human IgG or IgM.
  • the plate was washed four times with 300 pL/well Wash Buffer before 100 pL/we 11 undiluted tetramethylbenzidine (TMB, citrate buffer with hydrogen peroxide) substrate (Thermofisher, Switzerland) was added and incubated for 30 minutes at room temperature on a plate rotator (600 revolutions per minute, rpm). Before the absorbance was measured, 100 pL/well stop solution (0.25 M sulfuric acid) was added. The degree of colorimetric reaction was determined by absorption measurement at 450 nm with a microplate reader (Synergy HI, Microplate reader,
  • the plates were washed four times with wash buffer (300 m ⁇ /well) before either the anti -human IgM antibody-horseradish peroxidase conjugate or the anti human IgG antibody-horseradish peroxidase conjugate was added (100 m ⁇ /well).
  • the plate was incubated for two hours at 4-8°C. After washing the wells (4 x 300 m ⁇ /well), the substrate solution TMB was added (100 m ⁇ /well) and the plate incubated for further 30 minutes at 600 rpm and room temperature, protected from light.
  • the eluate was collected, a sample was taken from it and diluted 1:50 for analysis by the GM1 ELISA (Biihlmann Laboratories, Schonenbuch, Switzerland) as described above. In case of multiple flows through the column, the eluate was collected and added on top of the column again to allow another flow through the column, this was done up to 3x in selected experiments.
  • GM1 ELISA Biihlmann Laboratories, Schonenbuch, Switzerland
  • mice were four weeks old (12-15 g), had unlimited access to food and water, and were housed with a light/dark cycle of 12 h/12 h at constant temperature of 22 °C. Mice of either sex were sacrificed by CO2 inhalation. All experiments using mice were performed in accordance with a licence approved and granted by the United Kingdom Home Office.
  • mice were injected intraperitoneally (i.p.) with 4 mg anti-GMl ganglioside IgG. After 2 h, mice were injected intravenously (i.v.) into the tail vein with 2 mg of the glycopolymer 60 and after a further 30 min interval, 0.5 ml 100 % normal human serum (NHS) was delivered i.p. At 6 h, mice were asphyxiated with a rising concentration of CO2. Blood, diaphragm, and soleus muscles were collected for serum and immunohistochemical analysis.
  • tissue was washed and fixed with 4% paraformaldehyde in PBS immediately after anti-GMl IgG3 (DG2) incubation.
  • DG2 anti-GMl IgG3
  • Application of 0.1 M glycine for 10 min was performed to quench unreactive aldehyde groups.
  • Tissue was then incubated overnight at 4°C with anti-mouse IgG/M-FITC (1:300) in PBS. Tissue was rinsed in PBS and mounted in Citifluor mounting medium (Citifluor Products, UK).
  • the invented glycopolymers are based on a biodegradable poly-F-lysine backbone and carbohydrate epitopes that imitate the natural glycoepitope of the GMl-ganglioside.
  • the polymers 59- 97 are designed for a therapeutic application in patients, where pathogenic anti-GMl antibodies could be selectively neutralized and removed.
  • These anti-GMl IgG and IgM isotype autoantibodies are involved in autoimmune neurological diseases; their binding to GM1 in the peripheral nervous system triggers demyelination and neurodegeneration (see e.g., Willison, H.J. and N. Yuki, Peripheral neuropathies and anti-glycolipid antibodies. Brain, 2002.
  • 125(Pt 12): p. 2591-625 For the biological evaluation of the prepared glycopolymers, a collection of patient sera samples and an AMAN mouse model was used. Initially, the sera samples were tested positive for anti-GMl IgM or IgG antibodies. The synthetic glycopolymers 60-68 were tested for the inhibitory activity to block the binding of anti- GM1 antibodies to GM1 ganglioside. The different inhibitory activities were compared to the polymer carrying the natural GM1 glycoepitope 59 at two to four different concentrations in the competitive binding ELISA assay with a selection of patient sera.
  • FIG. 3A shows inhibition of binding of selected MMN patients’ sera anti-GMl IgM to GM1 ganglioside by exemplary polymeric compounds 59-68.
  • the inhibitory activity obtained during the biological characterization showed the different neutralization effects of the gly copolymers for anti-GMl IgM antibodies from different patients (FIG. 3A). This is probably due to interindividual differences of antibody characteristics (isotype, affinity, specificity, serum concentration, monoclonal/polyclonal, etc.) between the different patients.
  • FIG. 3B shows the inhibitory effect of the glycopolymers 59, 60, and 62 throughout the tested patient cohort (FIG. 3B), with polymer 60 showing most pronounced inhibition.
  • the data indicates that GM1 epitope mimics including a sialic acid group can provide potent inhibitory activity to neutralize anti-GMl IgM antibodies efficiently in serum of a large patient cohort.
  • the IC50 values of polymers 60, 69-97 were determined with anti-GMl antibody positive GBS patients’ sera.
  • the glycopolymer 60 exhibited an inhibitory activity in the picomolar to nanomolar range (Table 1). [0331]
  • FIG. 3C-3D shows the depletion of anti-GMl IgG (FIG. 3C) and anti-GMl IgM (FIG.
  • 3D antibodies from patient serum. 100-500 pi of patient serum were allowed to flow through (lx, top or 3x, bottom) a column containing 500 m ⁇ of GM1 mimetic (2) -functionalized Sepharose beads 98 (FIG. 3C) and 99 (FIG. 3D) by gravitational force. The eluate was collected for analysis of the amount of anti-GMl IgG or IgM antibodies remaining in the eluate after flowing through the column compared to the untreated serum before filtration. In FIG. 3D graph, the anti-GMl IgM absorbance of a healthy donor control sample is shown as a control (background).
  • FIG. 4 shows the effects of temperature on the inhibitory activity of exemplary polymeric compounds. Temperature dependent inhibition of anti- GM1 IgG to the GM1 ganglioside by polymer 62 and temperature independent inhibition by polymers 60 and 68 (***p> 0.001).
  • the polymer 60 and related polymers 61-97 are designed for a therapeutic application in peripheral neuropathies associated with anti-GMl IgG and IgM autoantibodies (e.g. AMAN,
  • FIG. 5 shows ex vivo inhibition of anti-GMl IgG binding and complement deposition to murine nerve terminals by polymer 60 (***p> 0.001).
  • Polymer 60 inhibited the binding of the mouse monoclonal anti-GMl IgG to the GM1 epitope on murine diaphragm tissue at ex vivo (FIG. 5).
  • the therapeutic utility of polymer 60 is further supported by inhibition of human complement deposition (C3c) at the target tissue.
  • C3c human complement deposition
  • FIG. 6A-6C shows inhibition of anti-GMl IgG binding to phrenic nerves by exemplary polymeric compound 60 in the in vivo nerve injury mouse model.
  • the i.v. injection of polymer 60 did reduce the binding of the anti-GMl IgG antibody (DG2) to the background signal (auto-fluorescence) in the target tissue, i.e., phrenic nerves in the diaphragm tissue (FIG. 6B, 6C).
  • a-Bungarotoxin (Btx) was used to stain the nicotinic acetylcholine receptors (nAChRs) and confirm the tissue samples were isolated and analyzed correctly (FIG.
  • GM1 gangliosides The enzyme required to produce GM1 gangliosides, GalNAcT, is driven by the Thyl promoter for neuronal membranes. Mice were bred with GM1 restricted neuronally to study selective GM1 binding along the axon (see e.g., Yao, D., et al. Journal of Neuroscience 2014, 34(3), 880-891).
  • TS triangularis stemi
  • FIG. 7B illustrates triangularis stemi muscle, dissected out of mouse rib cage, used for ex vivo investigation (created using BioRender).
  • DG2 anti-GMl antibody
  • Ex vivo preparations were incubated in Ringer’s solution with both DG2 and the GM1 gly copolymer or the control gly copolymer.
  • FIG. 8A shows a graph of in vitro dose response for binding and sequestering anti-GMl antibodies by the exemplary polymeric compound as compared to a control polymeric compound (control mimetic) that does not bind anti-GMl antibody.
  • FIG. 8B shows the related images of diaphragm tissue from neuronal GM1 -enriched mice.
  • Btx nicotinic acetylcholine receptor;
  • DG2 anti-GMl antibody.
  • Scale Bar 50 pm.***p ⁇ 0.001, **p ⁇ 0.01, one way ANOVA.
  • FIG. 9A shows a graph of DG2 antibody staining observed in the tissue for exemplary polymeric compound as compared to control polymeric compound (control mimetic) that does not bind anti-GMl antibody.
  • FIG. 9B shows images of triangularis stemi nerve-muscle tissue preparations from the neuronal GM1 -enriched mice stained with bungarotoxin to visualize the nerve-terminal and an anti-mouse IgG3 antibody to measure DG2 antibody.
  • Btx nicotinic acetylcholine receptor
  • DG2 anti-GMl antibody.
  • Scale Bar 20 pm;****p ⁇ 0.0001, unpaired two-tailed student t-test.

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Abstract

This disclosure provides glycan-conjugated compounds that specifically bind to anti -GM1 autoantibodies. These glycan-conjugated compounds can include a polymer backbone or support configured to display multiple glycan groups that are designed to mimic the natural GM1 epitope. The glycan-conjugated compounds can also be configured on a solid support. The linked glycan groups can be a novel analogue of the GM1 epitope. When several such glycan groups are configured on a polymeric backbone, the resulting compound provides for potent and specific binding to anti-GM1 IgG and IgM isotype autoantibodies in patient sera. This disclosure thus provides glycan-containing compounds of formula (I) that mimic the natural GM1 epitope, and therapeutically useful polymers that include a multitude of such ligands, designed to bind anti-GM1 antibodies in vitro for diagnostic use, and to sequester and eliminate anti-GM1 antibodies in vivo or extracorporeal (ex vivo) for the treatment anti-GM1 autoantibody related neuropathies.

Description

ANTI-GM1 ANTIBODY BINDING COMPOUNDS
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Nos. 63/177,263, filed April 20, 2021; and 63/191,024, filed May 20, 2021; both of which are incorporated by reference in their entirety.
2. BACKGROUND OF THE INVENTION
2.1. Field of the invention.
[0002] The invention relates to compounds including carbohydrate ligands that bind to antibodies (IgG and IgM isotype) against the GM1 ganglioside, and to their use in diagnostic and therapeutic applications related to anti-GMl antibody-associated neuropathies, such as Guillain-Barre-Syndrome (GBS) and multifocal motor neuropathy (MMN).
2.2. Description of related art.
[0003] Various neurological diseases are associated with the presence or increased levels of anti- glycan antibodies. Anti -ganglioside antibodies, particularly anti-GMl antibodies have been detected in a various peripheral neuropathies, including variants of GBS such as acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) and the pharyngeal -cervical-brachial variant of GBS, and the chronic multifocal motor neuropathy MMN. GBS has an incidence of approximately 1-2 in 100,000 people whereas the prevalence of MNN is approximately 1 in 100,000 people.
[0004] GM1 belongs to the family of gangliosides (glycosphingolipids), it is composed of a ceramide tail and a pentasaccharide head group containing one sialic acid (N-acetylneuraminic acid, Neu5Ac). GM1 occurs widely in human tissues, where it exhibits a variety of essential functions, both in the plasma membrane and intracellular loci. GM1 is typically located in membrane rafts and functionally prominent in lipid microdomains. GM1 interacts with proteins that modulate mechanisms such as ion transport (transport of Ca2+, Na+, and K+ via ion channels and ion exchangers), neuronal differentiation, signaling via G protein-coupled receptors (GPCRs), immune system reactivities (effector T cells), and neuroprotective signaling. The latter occurs through intimate association with neurotrophin receptors, which has relevance to the etiopathogenesis of neurodegenerative diseases and potential therapies.
[0005] There is strong evidence from ex vivo studies with patients’ sera, animal models and clinical studies that anti-GMl antibodies are directly and indirectly involved in immune-mediated attack towards the nervous system. The autoantibodies bind to the GM1 ganglioside, disrupt the signalosome systems directly (e.g. ion transport) or cause complement system -mediated axonal damage. 3. SUMMARY OF THE INVENTION
[0006] This disclosure provides glycan-conjugated compounds that specifically bind to anti-GMl autoantibodies. These glycan-conjugated compounds can be conjugated to a polymeric backbone and/or support configured to display multiple glycan groups and designed to mimic the natural GM1 epitope. The glycan-conjugated compounds can also be configured on a polymeric support such as a Sepharose/agarose bead suitable for purification or separation of biological samples. In some embodiments, the linked glycan groups are selected from a series of novel analogues of the GM1 epitope (FIG. 1). When several such glycan groups are presented on a polymeric backbone the resulting polymeric compound provides for potent and specific binding to anti-GMl IgG and IgM isotype autoantibodies in patient sera. This disclosure thus provides glycan-containing compounds of formula (I) that mimic the natural GM1 epitope, and therapeutically useful polymers that include a multitude of such ligands, designed to bind anti-GMl antibodies in vitro for diagnostic use, and more importantly designed to sequester and eliminate anti-GMl antibodies in vivo or extracorporeal (ex vivo) for the treatment anti-GMl autoantibody related neuropathies, such as AMAN, AMSAN, and MMN.
4. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0007] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:
[0008] FIG. 1. Representative structures of the natural GM1 epitope (compound 1) and a series of GM1 glyco-mimetics with modifications on the GM1 core structure, such as: a) replacement of the reducing end glucose (Part-I) with a tyramine moiety (compound 2-7); b) replacement of the Neu5Ac (Part-II) with 2-(3 -cyclohexyl -propanoic acid), 2-(3 -phenyl-propanoic acid), and 2-propanoic acid (compound 3-9).
[0009] FIGs. 2A-2C. Representative retrosynthesis of the exemplary GM1 ligand compounds including exemplary GM1 glyco-mimetics. Sialic acid is first attached to the 3 -position of galactosides (23a-b) and the propionates are introduced via tin-mediated alkylation reactions (®29a- c, 34a-b, 37a-b) (FIG. 2C). Two different Gal-GalN building blocks (13, 17) are prepared from monosaccharide donors (10, 14) and acceptors (11, 15) and used in the subsequent glycosylation reactions (FIG. 2B). The key step of the GM1 synthesis is the coupling of Gal-GalN disaccharide fragments (13, 17) to selectively protected di- or tri-saccharide acceptors (29a-c, 34a-b, 37a-b) via both the conventional and pre -activation glycosylation conditions (FIG. 2A).
[0010] FIGs. 3A-3B show the inhibitory activity of exemplary polymeric compounds. FIG. 3A shows inhibition of binding of selected MMN patients’ sera anti-GMl IgM to GM1 ganglioside by exemplary polymeric compounds 59-68. FIG. 3B is a plot of inhibitory activity of polymeric compounds 59, 60, and 62 to block the binding of MMN patients’ sera anti-GMl IgM to GM1 ganglioside (n = 23 MMN patients, ***p> 0.001, **** p>0.00001).
[0011] FIGs. 3C-3D shows the depletion of anti-GMl IgG (FIG. 3C) and anti-GMl IgM (FIG. 3D) antibodies from patient serum using GM1 mimetic (2)-functionalized Sepharose beads.
[0012] FIG. 4 shows the effects of temperature on the inhibitory activity of exemplary polymeric compounds. Temperature dependent inhibition of anti-GMl IgG to the GM1 ganglioside by polymer 62 and temperature independent inhibition by polymers 60 and 68 (***p> 0.001).
[0013] FIG. 5. Ex vivo inhibition of anti-GMl IgG binding and complement deposition to murine nerve terminals by polymer 60 (***p> 0.001).
[0014] FIG. 6A-6C shows inhibition of anti-GMl IgG binding to phrenic nerves by exemplary polymeric compound in an in vivo nerve injury mouse model of AMAN.
[0015] FIGs. 7A-7B depicts details of tissue preparations from neuronal GM1 -enriched mice that were used to assess binding in vitro and ex vivo of an exemplary polymeric compound (polymer 60; “mimetic 1” = GM1 mimetic (2)) versus a control polymeric compound (control mimetic). FIG. 7A depicts sections of murine diaphragm used for in vitro investigation. FIG. 7B illustrates triangularis stemi nerve-muscle preparations, dissected out of mouse rib cage, used for ex vivo investigation (created using BioRender).
[0016] FIGs. 8A-8B illustrates that an exemplary polymeric compound (polymer 60; “mimetic 1” = GM1 mimetic (2)) fully sequesters anti-GMl IgG antibody DG2 in vitro. FIG. 8A shows a graph of in vitro dose response for binding and sequestering anti-GMl antibodies by an exemplary polymeric compound as compared to a control polymeric compound (control mimetic) that does not bind anti- GMl antibody. FIG. 8B shows the related images of diaphragm tissue from neuronal GM1 -enriched mice. Btx=nicotinic acetylcholine receptor; DG2=anti-GMl IgG antibody. Scale Bar = 50 pm.***p < 0.001, **p < 0.01, one-way ANOVA.
[0017] FIGs. 9A-B show that exemplary polymeric compound (polymer 60; “mimetic 1” = GM1 mimetic (2)) reduces anti-GMl antibody binding ex vivo. FIG. 9A shows a graph of DG2 antibody staining observed in the triangularis stemi nerve-muscle tissue for exemplary polymeric compound as compared to control polymeric compound (control mimetic) that does not bind anti-GMl antibody. FIG. 9B. shows related images of tissue preparations from neuronal GM1 -enriched mice stained with bungarotoxin to visualize the nerve-terminal and an anti-mouse IgG3 antibody to measure DG2 antibody. Btx=nicotinic acetylcholine receptor; DG2=anti-GMl IgG antibody. Scale Bar = 20 pm;****p < 0.0001, unpaired two-tailed student t-test. 5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Glycan-Containing Compounds and Conjugates
[0018] As summarized above, this disclosure provides glycan-containing compounds and conjugates. In some embodiments, the compounds and conjugates include several glycan groups linked to a moiety of interest (e.g., a polymer, carrier or support). In some embodiments, the compounds include one or more glycan groups linked to a chemoselective ligation group, or a synthetic precursor thereof, suitable for use in preparation of a compound or conjugate including a linked moiety of interest. [0019] The glycan groups are designed to mimic the pentasaccharide head group of GM1 and incorporate an aryl or heteroaryl linking moiety that provides connection via a linker to the moiety of interest with a configuration that provides potent binding to the anti-GMl autoantibodies, especially anti-GMl autoantibodies having neuropathic potential. We have demonstrated that when several of such glycan group-containing compounds are configured on a suitable construct, they can provide potent binding and blocking of the disease-causing target anti-GMl autoantibodies. A variety of constructs can be used as a basis for multivalent display of the glycan groups to provide for desirable binding and blocking activity against target anti-GMl autoantibodies, and are meant to be encompassed by the compounds and conjugates of this disclosure. In some embodiments, the construct is a polymer that provides multiple points of attachment for the glycan groups. In some embodiments, the construct is a support, such as a bead or planar solid support suitable for multivalent display of the glycan groups. The particular constructs, compounds and conjugates of this disclosure can be selected to provide desirable binding and blocking activity against target anti-GMl autoantibodies depending on the particular biological sample or system in which it is used.
[0020] We have demonstrated that exemplary anti-GMl antibody-binding polymeric compounds and conjugates of this disclosure bind and block target anti-GMl antibodies in a biological system, e.g., the human serum of a patient (ex vivo) or in vivo in a mouse model for AMAN. Accordingly, the anti- GMl antibody-binding compounds and conjugates of this disclosure are useful in anti-GMl antibody associated neuropathy applications, including therapeutic and diagnostic applications.
5.1.1. Linked Glycans
[0021] As summarized above, the anti-GMl antibody-binding compounds of this disclosure can include one or more, two or more, or several glycan groups that mimic GM1, and which can be linked via a chemoselective ligation group to a moiety of interest. It is understood that the anti-GMl antibody-binding compounds can be conjugated to moiety of interest suitable for an in vivo, or ex vivo therapeutic or diagnostic use.
[0022] GM1 is a glycosphingolipid that belongs to the family of gangliosides and is composed of a ceramide tail and a penta-saccharide head group containing the sialic acid, N-acetylneuraminic acid (Neu5Ac). Particular disease-causing anti-GMl autoantibodies are associated with various neuropathy indications, such as AMAN, AMSAN, and MMN.
[0023] We developed compounds that include a modified tri- or tetra-saccharide GM1 head group that incorporates an aryl or heteroaryl group to replace one of the naturally occurring monosaccharides of GM1, and provide for a suitable attachment point to a linker. This glycan group provides for desirable high affinity and selective binding to target anti-GMl autoantibodies.
[0024] In some embodiments, the glycan group has a tetra-saccharide portion of the head group that includes a sialic acid group. In some embodiments, the sialic acid group is the naturally occurring N- acetylneuraminic acid, or an analog such as N-g 1 y co hi n c uram i n i c acid. We have further demonstrated that glycan groups with a modified tri-saccharide having a suitable sialic acid group replacement that is an optionally substituted carboxymethyl group (e.g., a HO2C-CH2- group where the methylene carbon is further substituted) can provide for desirable high affinity binding to target anti-GMl autoantibodies.
[0025] In some embodiments, the glycan group of the anti-GMl antibody-binding compound is of formula (XI):
Figure imgf000006_0001
wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group;
Z1 is -0-, -S-, -NR2- or -C(R2)2-, wherein each R2 is independently selected from H, (C1-C4)- alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5;
Ar is an optionally substituted aryl or optionally substituted heteroaryl; and L1 is a linker (e.g., as described herein).
[0026] In some embodiments of formula (XI), Ar is optionally substituted aryl or optionally substituted heteroaryl.
[0027] In some embodiments of formula (XI), Ar is an optionally substituted 5-membered monocyclic heteroaryl group. In some embodiments of formula (XI), Ar is an optionally substituted 6- membered monocyclic aryl or heteroaryl group. The Ar group linking moiety of formula (XI) can be an optionally substituted multicyclic aryl or multicyclic heteroaryl group, such as an optionally substituted bicyclic aryl or bicyclic heteroaryl group. In some embodiments of formula (XI), Ar is an optionally substituted fused bicyclic group. In some embodiments of formula (XI), Ar is an optionally substituted bicyclic group comprising two aryl and/or heteroaryl monocyclic rings connected via a covalent bond. In some embodiments of formula (XI), Ar is an optionally substituted bicyclic aryl or bicyclic heteroaryl group having two 6-membered rings. In some embodiments of formula (XI), Ar is an optionally substituted bicyclic aryl or bicyclic heteroaryl group having one 6-membered ring that is connected via a covalent bond or fused to a 5-membered ring.
[0028] In some embodiments of formula (XI), each Ar is independently selected from optionally substituted phenyl, optionally substituted pyridyl, optionally substituted biphenyl, optionally substituted naphthalene, and optionally substituted quinoline. In some embodiments of formula (XI), Ar is substituted with one or substituents selected from OH, halogen, (Ci-Cg)-alkyl, optionally substituted (Ci-Cg)-alkyl, (Ci-Cg)-alkoxy, and optionally substituted (Ci-Cg)-alkoxy. In some embodiments of formula (XI), Ar is optionally substituted 1,4-phenylene, optionally substituted 1,3- phenylene, or optionally substituted 2,5-pyridylene.
[0029] In some embodiments of formula (XI), the glycan group of the anti-GMl antibody-binding compound is of formula (XII):
Figure imgf000007_0001
or a pharmaceutically acceptable salt thereof, wherein: q is 0 to 4; and each R11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
[0030] In some embodiments of formula (XI)-(XII), R1 is a sialic acid group. Sialic acid group refers to a monosaccharide of the sialic acid class of alpha-keto acid sugars having a nine-carbon backbone. The sialic acid can be naturally occurring, e.g., as terminating branches of N-glycans, O-glycans, and glycosphingolipids (gangliosides). In some embodiments, the sialic acid group is a N- and O- substituted derivative of neuraminic acid. In some embodiments, the sialic acid group is N- acetylneuraminic acid. In some embodiments, the sialic acid group is N- glycolylneuraminic acid. [0031] In some embodiments of formula (XI)-(XII), R1 is optionally substituted carboxymethyl group, e.g., a HO2C-CH2- group wherein the methylene carbon is further optionally substituted. [0032] In some embodiments of formula (XI)-(XII), R1 is selected from formula (XHIa) and (Xlllb):
Figure imgf000008_0001
(XHIa) (XHIb) or a pharmaceutically acceptable salt thereof, wherein:
R21 is optionally substituted (Ci-C3)-alkyl; and
R22 and R23 are independently selected from H, optionally substituted (Ci-C3)-alkyl, optionally substituted aryl-(Ci-C3)-alkylene-, optionally substituted heteroaryl-(Ci-C3)-alkylene-, optionally substituted cycloalkyl-(Ci-C3)-alkylene-, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl.
[0033] In some embodiments of formula (XHIa), R21 is methyl. In some embodiments of formula (XHIa), R21 is hydroxymethyl (HO-CH2-).
[0034] In some embodiments of formula (XI)-(XII), R1 is of formula (XHIa), wherein R21 is methyl:
Figure imgf000008_0002
or a pharmaceutically acceptable salt thereof.
[0035] In some embodiments of formula (XI)-(XII), R1 is of formula (XHIa), wherein R21 is hydroxymethyl:
Figure imgf000008_0003
or a pharmaceutically acceptable salt thereof.
[0036] In some embodiments of formula (XHIb), R22 is H and R23 is not H.
[0037] In some embodiments of formula (XHIb), R22 is H and R23 is optionally substituted (C1-C3)- alkyl. In some embodiments of formula (XHIb), R22 is H and R23 is optionally substituted aryl-(Ci- C3)-alkylene-. In some embodiments of formula (XHIb), R22 is H and R23 is optionally substituted phenyl-(Ci-C3)-alkylene-. In some embodiments of formula (XHIb), R22 is H and R23 is optionally substituted benzyl. In some embodiments of formula (XHIb), R22 is H and R23 is optionally substituted cyclohexyl-(Ci-C3)-alkylene-. In some embodiments of formula (XHIb), R22 is H and R23 is optionally substituted cyclohexyl-methyl-.
[0038] In some embodiments of formula (XI)-(XII), R1 is of (XHIb) and selected from:
Figure imgf000009_0001
or a pharmaceutically acceptable salt thereof.
[0039] In some embodiments of formula (XHIb), R1 is selected from:
Figure imgf000009_0002
or a pharmaceutically acceptable salt thereof.
[0040] In some embodiments of formula (XI)-(XII), Z1 is -0-. In some embodiments of formula (XI)-(XII), Z1 is -S-.
[0041] In some embodiments of formula (XI)-(XII), Z1 is -NR2-, where R2 is selected from H, (Ci- C4) -alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5. In some embodiments of formula (XI)-(XII), Z1 is -NR2-, where R2 is selected from H, and (Ci-C4)-alkyl. In some embodiments, Z1 is -NMe-.
[0042] In some embodiments of formula (XI)-(XII), Z1 is -NH-.
[0043] In some embodiments of formula (XI)-(XII), Z1 is -C(R2)2-. In some embodiments of formula (XI)-(XII), Z1 is -CH2-.
[0044] Exemplary glycan groups include, but are not limited to,
Figure imgf000009_0003
PCT/IB2022/000224
Figure imgf000010_0001
Figure imgf000011_0001
or a pharmaceutically acceptable salt thereof.
[0045] In some embodiments of formula (XI) to (XII), the linked glycan is
Figure imgf000011_0002
or a pharmaceutically acceptable salt thereof.
[0046] In some embodiments of formula (XI) to (XII), the linked glycan is
Figure imgf000011_0003
or a pharmaceutically acceptable salt thereof.
[0047] In some embodiments of formula (XI) to (XII), the linked glycan is
Figure imgf000012_0001
or a pharmaceutically acceptable salt thereof.
5.1.2. Linkers
[0048] Any convenient linker (L1) can be incorporated into the glycans of formula (XI)-(XII) to connect the glycan group of this disclosure to a chemoselective ligation group or other moiety of interest (e.g., Y of formula (I), or P of formula (III), or Z2 of formula (V), as described herein). The terms “linker”, “linking moiety” and “linking group” are used interchangeably and refer to a linking moiety that covalently connects two or more moieties or compounds, such as glycans and other moieties of interest.
[0049] In some embodiments, the linker is divalent and connects a single glycan group to a moiety of interest. In certain embodiments, the linker is a branched linking group that is trivalent or of a higher multivalency, and is capable of linking two, three or more glycan groups to a moiety of interest.
[0050] In some embodiments, the linker that connects the two or more moieties has a linear or branched backbone of 500 atoms or less (such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less) in length, e.g., as measured between the two or more moieties.
[0051] A linking moiety may be a covalent bond that connects two groups or a linear or branched chain of between 1 and 500 atoms in length, for example of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, 200, 300, 400 or 500 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom. In certain cases, one, two, three, four, five or more, ten or more, or even more carbon atoms of a linker backbone may be optionally substituted with heteroatoms, e.g., sulfur, nitrogen or oxygen heteroatom. In certain instances, when the linker includes an ethylene glycol or polyethylene glycol) group, every third atom of that segment of the linker hydrocarbon backbone is substituted with an oxygen. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups. A linker may further include, without limitations, one or more of the following: ether, thioether, disulfide, amide, carbonate, carbamate, tertiary amine, or alkyl, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), nbutyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle, a cycloalkyl group or a heterocycle group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
[0052] In some embodiments, a “linker” or linking moiety, or a portion thereof, is derived from a molecule with two reactive termini (e.g., a bifunctional linker), one for conjugation to a moiety of interest (e.g., as described herein) and the other for linkage to a glycan group.
[0053] In certain embodiments of the formula described herein, the linker L1 includes one or more straight or branched-chain carbon moieties and/or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CH2CH2O- or -OCH2CH2-), and combinations thereof, optionally connected via one or more linkages. In certain embodiments, these linkers optionally have amide linkages, urea or thiourea linkages, carbamate linkages, ester linkages, amino linkages, ether linkages, thioether linkages, sulfhydryl linkages, or other hetero-functional linkages.
[0054] In certain embodiments, the linker comprises one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof. In certain embodiments, the linker comprises one or more of an ether bond, thioether bond, amine bond, amide bond, carbon-carbon bond, carbon- nitrogen bond, carbon-oxygen bond, carbon-sulfur bond, and combinations thereof. In certain embodiments, the linker comprises a linear structure. In certain embodiments, the linker comprises a branched structure. In certain embodiments, the linker comprises a cyclic structure.
[0055] In certain embodiments, L is a linker between about 5 A and about 500 A. In certain embodiments, L is between about 10 A and about 400 A. In certain embodiments, L is between about 10 A and about 300 A. In certain embodiments, L is between about 10 A and about 200 A. In certain embodiments, L is between about 10 A and about 100 A.
[0056] It is understood that the linker may be considered as connecting directly to the Ar group of a glycan compound (e.g., Ar of Formula (XI)-(XII) as described herein). It is understood that the linker may encompass one or more linking functional groups that are the residual product of a coupling reaction between two chemoselective ligation groups (e.g., as described herein). Residual linking functional groups derived from any of the conjugation chemistries described herein can be incorporated into the linkers of the compounds and conjugates of this disclosure.
[0057] In some embodiments of Formula (XI) to (XII), L1 is linear linker comprising one or more linking moieties independently selected from -Ci-6-alkylene-, -NHCO-Ci-6-alkylene-, -CONH-C1-6- alkylene-, -NH Ci-6-alkylene-, -NHCONH-Ci-6-alkylene-, - NHCSNH-Ci-6-alkylene-, -C1-6- alkylene-NHCO, -Ci-6-alkylene-CONH-, -Ci-6-alkylene-NH-, -Ci-6-alkylene-NHCONH-, -C1-6- alkylene-NHC SNH-, -0(CH2)P-, -(OCH2CH2)P-, -NHCO-, -CONH-, -NHSO2-, -SO2NH-, -CO-, -SO2-, -0-, — S — , pyrrolidine-2,5 -dione, -NH-, and -NMe-, wherein p is 1 to 10. [0058] In some embodiments of Formula (XI) to (XII), L1 is -Zu-(Ci-C6)-alkyl-Z12-(Ci-C6)-alkyl-, where: Z11 is absent, CO, C(=0)NH, S02NH, O, S, NH, -NHC(=0)-, -NHC(=0)NH-, or - NHC(=S)NH-; and Z12 is absent, -NHC(=0)-, C(=0)NH, -NHC(=NH)-, S02NH, O, S, or NH.
[0059] In some embodiments of Formula (XI) to (XII), L1 is -(CH2)qNH(C=0)(CH2)rS-CH2-(C=0)- connecting Ar to an amine-containing group of the moiety of interest Y, where q and r are independently 1 to 10, such as 1 to 6, or 1 to 3.
[0060] In some embodiments of Formula (XI) to (XII), L1 is -(CH2)2NH(C=0)(CH2)3S-CH2-(C=0)- connecting Ar to an amine -containing group, e.g., an amine -containing repeat unit of a polypeptide backbone.
[0061] As summarized above, the glycan groups of this disclosure can be linked to a moiety of interest (Y) via a linker (L1). In some embodiments, the linkage of a glycan-linker compound to a moiety of interest (Y) is achieved via a conjugation or ligation reaction between two compatible functional groups, i.e., a first chemoselective ligation group that is incorporated into the linker of a glycan-linker compound, and a compatible chemoselective ligation group of the moiety of interest.
5.1.3. Chemoselective ligation groups
[0062] A variety of chemoselective ligation groups, or synthetic precursors thereof, can be incorporated into the glycan-linker compounds of this disclosure. A chemoselective ligation group is a group having a reactive functionality or functional group capable of conjugation to a compatible group of a second moiety. For example, chemoselective ligation groups (or precursors thereof) may be one of a pair of groups associated with a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Pictet- Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g., thiol-maleimide, thiol- haloacetamide or alkyne hydrothiolation), amine-active ester amido bond coupling, reductive amination, dialkyl squarate chemistry, etc..
[0063] Chemoselective ligation groups that may be utilized in linking a glycan-linker to a moiety of interest, include, but are not limited to, amine (e.g., a N-terminal amine or a lysine side chain amine group of a polypeptide), azide, aryl azide, alkynyl (e.g., ethynyl or cyclooctyne or derivative), active ester (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester or pentafluorophenyl (PFP) ester or thioester), haloacetamide (e.g., chloroacetamide, iodoacetamide or bromoacetamide), chloroacetyl, bromoacetyl, hydrazide, maleimide, vinyl sulfone, 2-sulfonyl pyridine, cyano-alkyne, thiol (e.g., a cysteine residue), disulfide or protected thiol, isocyanate, isothiocyanate, aldehyde, ketone, aminooxy or alkoxyamine, hydrazide, hydroxylamino, phosphine, HIPS hydrazinyl-indolyl group, or aza-HIPS hydrazinyl-pyrrolo-pyridinyl group, tetrazine, cyclooctene, squarate, and the like.
[0064] In some instances, a chemoselective ligation group is capable of spontaneous conjugation to a compatible chemical group when the two groups come into contact under suitable conditions (e.g., copper free Click chemistry conditions). In some instances, the chemoselective ligation group is capable of conjugation to a compatible chemical group when the two groups come into contact in the presence of a catalyst or other reagent (e.g., copper catalyzed Click chemistry conditions).
[0065] In some embodiments, the chemoselective ligation group is a photoactive ligation group. For example, upon irradiation with ultraviolet light, a diazirine group can form reactive carbenes, which can insert into C-H, N-H, and O-H bonds of a second moiety.
[0066] In some embodiments, Z2 is a precursor of the reactive functionality or function group capable of conjugation to a compatible group of a second moiety. For example, a carboxylic acid is a precursor of an active ester chemoselective ligation group.
[0067] In certain embodiments of formula (V), Z2 is a reactive moiety capable forming a covalent bond to a polypeptide (e.g., with an amino acid side chain of a polypeptide having a compatible reactive group). The reactive moiety can be referred to as a chemoselective ligation group.
[0068] In certain embodiments of formula (V), Z2 is a thiol-reactive chemoselective ligation group. In some embodiments, Z2 can produce a residual moiety (Z3) that results from the covalent linkage of a thiol with a thiol-reactive chemoselective ligation group of a polymer backbone residue, e.g., a polypeptide having side chain groups that are modified to include a thiol-reactive chemoselective ligation group, such as a haloacetamide.
[0069] In certain embodiments of formula (V), Z2 is an amine group, that can be coupled with an amino-reactive chemoselective ligation group of a moiety of interest. In some embodiments, Z2 can produce a residual moiety (Z3) resulting from the covalent linkage of an amine and an amine-reactive chemoselective ligation group (e.g., an active ester group) of a moiety of interest, e.g., Z3 is an amide linkage.
5.1.4. Glycan-Linker Compounds
[0070] As summarized above, the glycan groups of this disclosure (e.g., of formula (XI)-(XII)) can be linked to a moiety of interest (Y) via a linker (L1). Accordingly, another aspect of this disclosure is glycan-linker compounds that include a chemoselective ligation group useful in attaching the linked glycans to a variety of constructs for the preparation of compounds and conjugates of this disclosure. [0071] In some embodiments, the glycan-linker compound includes a glycan of formula (XI)-(XII), and is of formula (V):
Figure imgf000015_0001
or a salt thereof, wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group.
Z1 is -0-, -S-, -NR2- or -C(R2)2-, wherein each R2 is independently selected from H, (C1-C4)- alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5;
Ar is optionally substituted aryl or optionally substituted heteroaryl (e.g., monocyclic aryl or heteroaryl, or bicyclic aryl or heteroaryl);
L1 is a linker;
Z2 is a chemoselective ligation group, or a precursor thereof.
[0072] Each of the embodiments of the glycan-linker groups of formula (XI)-(XII) described above can be incorporated into a glycan-linker compound of formula (V). Thus, every embodiment of R1,
Z1, Ar and L1 described above is meant to be encompassed by formula (V).
[0073] In some embodiments of formula (V), L1 is linear linker comprising one or more linking moieties independently selected from -Ci-6-alkylene-, -NHCO-Ci-6-alkylene-, -CONH-C1-6- alkylene-, -NH Ci-6-alkylene-, -NHCONH-Ci-6-alkylene-, - NHCSNH-Ci-6-alkylene-, -C1-6- alkylene-NHCO, -Ci-6-alkylene-CONH-, -Ci-6-alkylene-NH-, -Ci-6-alkylene-NHCONH-, -C1-6- alkylene-NHC SNH-, -0(CH2)p- -(OCH2CH2)p- -NHCO-, -CONH-, -NHS02-, -S02NH-, -CO-, — S02 — , -0-, — S — , pyrrolidine-2,5 -dione, -NH-, and -NMe-, wherein p is 1 to 10.
[0074] In some embodiments of formula (V), Z2 is selected from -NH2, -SH, -N3, alkyne, active ester, and maleimide. In some embodiments of formula (V), Z2 is amine (e.g., -NH2). In some embodiments of formula (V), Z2 is thiol (e.g., -SH).
[0075] In some embodiments of formula (V), -L'-Z2 is -(Ci-C6)-alkyl-NH2. In some embodiments of formula (V), -L'-Z2 is -(CH2)2NH2. In some embodiments, e.g., when -L'-Z2 is -(Ci-C6)-alkyl-NH2, the linker of the compound can be extended to incorporate a longer L1 and/or an alternative Z2. For example, a bifunctional linker or reagent can be attached to the terminal Z2 chemoselective ligation group, to produce another glycan-linker compound of formula (V). Exemplary bifunctional linkers or reagents which can be utilized to extend or modify the glycan-linker compounds of formula (V) include, but are not limited to, Traut’s reagent and g-thiobutyrolactone.
[0076] In some embodiments of Formula (V), L1 is -Zu-(Ci-C6)-alkyl-Z12-(Ci-C6)-alkyl-, where: Z11 is absent, CO, C(=0)NH, S02NH, O, S, NH, -NHC(=0)-, -NHC(=0)NH-, or -NHC(=S)NH-; and Z12 is absent, -NHC(=0)-, C(=0)NH, -NHC(=NH)-, S02NH, O, S, orNH.
[0077] In some embodiments of formula (V), -L'-Z2 is -(Ci-C6)-alkyl-amido-(Ci-C6)-alkyl-SH. In some embodiments of formula (V), -L'-Z2 is -(CH2)2NHCO(CH2)3SH.
[0078] In some embodiments of formula (V), the compound is selected from:
Figure imgf000017_0001
Figure imgf000018_0001
or a salt thereof, wherein:
-L1- is -Z11-(Ci-C6)-alkyl-Z12-(Ci-C6)-alkyl-;
Z11 is absent, CO, C(=0)NH, SO2NH, O, S, NH, -NHC(=0)-, -NHC(=0)NH-, or - NHC(=S)NH-;
Z12 is absent, -NHC(=0)-, -C(=0)NH-, -NHC(=NH)-, -SO2NH-, -0-, -S-, or -NH-; and Z2 is selected from -NH2, -SH, -N3, alkyne, active ester, and maleimide. In some embodiments of these compounds, Z2 is NH2. In some embodiments of these compounds, Z2 is SH.
In some embodiments of these compounds, -L'-Z2 is -(Ci-C6)-alkyl-amido-(Ci-C6)-alkyl-SH. In some embodiments of these compounds, -L'-Z2 is -(CH2)2NHCO(CH2)3SH.
[0079] In some embodiments of formula (V), the compound is
Figure imgf000018_0002
or a salt thereof.
[0080] In some embodiments of formula (V), the compound is
Figure imgf000019_0001
or a salt thereof.
[0081] In some embodiments of formula (V), the compound is
Figure imgf000019_0002
or a salt thereof.
[0082] In some embodiments of formula (V), the compound is
Figure imgf000019_0003
or a salt thereof.
[0083] In some embodiments of formula (V), the compound is
Figure imgf000019_0004
or a salt thereof. [0084] In some embodiments of formula (V), the compound is
Figure imgf000020_0001
or a salt thereof.
5.1.5. Moieties of Interest (Y)
[0085] Aspects of this disclosure include glycan-containing compounds and conjugates that include a glycan group (e.g., of formula (XI)-(XII) described herein) linked to a moiety of interest. As summarized above, the linkage of glycan-linker compounds to a moiety of interest (Y) can be achieved via a chemoselective ligation (e.g., as described herein).
[0086] The compounds of this disclosure can be referred to interchangeably as conjugates, e.g., when the moiety of interest (Y) is a molecule or construct such as, a polymer, carrier biomolecule or support. Such conjugates can be prepared by coupling of a chemoselective ligation group on a glycan- linker compound with a compatible reactive group of Y. The compatible reactive group can be introduced into a moiety of interest by modification prior to conjugation, or can be a group already present in Y. Alternatively, the conjugates of this disclosure can be prepared by incorporating a linker into a moiety of interest to which a glycan group can be attached.
5.1.5.1 Polymers
[0087] In some embodiments, the moiety of interest Y is a polymer. A variety of polymers can be utilized that have a polymer backbone composed of repeat units providing for attachment of a multitude of linked glycan compounds. The polymer can be a homopolymer. The polymer can be a heteropolymer, such as a block copolymer or a random copolymer. The polymer can be prepared in a stepwise fashion and have a discrete or defined length and sequence. Alternatively, the polymer can be prepared via a bulk polymerization method.
[0088] In some embodiments, the polymer is pharmaceutically acceptable, e.g., a biocompatible polymer suitable for therapeutic use in a polymeric compound of this disclosure. In some embodiments, the polymer is one suitable for in vivo applications where the polymer-glycan conjugate can be administered as a therapeutic agent to a subject in need thereof. In some embodiments, the polymer is one suitable for ex vivo applications, e.g., where the polymer-glycan conjugate can be used extracorporeally as part of an immune -adsorption support or apparatus configured to remove target autoantibodies from a biological sample.
[0089] Polymers of interest include, but are not limited to, amino acid polymers (referred to interchangeably as polypeptides), acrylic acid or methacrylic acid polymers or copolymers (e.g., polyacrylate, poly(meth)acrylate or copolymer thereof), N-vinyl-2-pyrrolidone-vinylalcohol copolymers, polysaccharide polymers, agarose, cellulose, chitosan polymers, and polyphosphazene polymers.
[0090] In a preferred embodiment, the polymer is an amino acid polymer. The amino acid polymer can be composed of any convenient amino acid residues, including naturally occurring or non- naturally occurring amino acid residues, and including alpha- or beta- or delta- or gamma-amino acid residues, or any combinations thereof. In a preferred embodiment, the polymer is an alpha-amino acid polymer.
[0091] Amino acids, which can be incorporated into a polymer for use in the polymeric compounds of this disclosure include those having a side chain moiety suitable for conjugation to a glycan-linker compound (e.g., as described herein), such as amino acids selected from lysine, ornithine, glutamic acid, aspartic acid, serine and cysteine. In some embodiments, the side chain moiety of the amino acid can be modified (before or after polymerization) to incorporate a chemoselective ligation group. In some embodiments, the amino acid polymer includes residues that are selected to impart desirable properties (e.g., physical or optical properties such as hydrophilicity, hydrophobicity, charge, pi, UV absorbance, etc.) on the resulting polymeric compound. Amino acids of interest which may be incorporated into the polymer include, but are not limited to, lysine, ornithine, glutamic acid, aspartic acid, cysteine, alanine, glycine, serine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine, and the like.
[0092] The amino acid polymer can be linear, branched, hyperbranched or dendritic, as described by Z. Kadlecova et al, Biomacromolecules 2012, 13: 3127-3137.
[0093] In a preferred embodiment, the polymer backbone is an alpha-amino acid polymer, and the polymer is composed of amino acid resides selected from lysine, ornithine, glutamic acid, aspartic acid, and serine.
[0094] In a very preferred embodiment, the polymer is poly-lysine. In some embodiments, the polymer is poly-Z -lysine. In some embodiments, the polymer is poly-D-lysine. In some embodiments, the polymer is a co-polymer of lysine residues and one or more additional amino acid residues (e.g., as described herein).
[0095] In some embodiments, the polymer is poly-aspartic acid or poly-glutamic acid. In some embodiments, the polymer is poly-Z- aspartic acid. In some embodiments, the polymer is a co polymer of aspartic acid or glutamic acid residues and one or more additional amino acid residues (e.g., as described herein). The side chains of a poly-aspartic acid or poly-glutamic acid polymer can be functionalized or derivatized by esterification and/or amide bond formation.
[0096] In some embodiments, the molecular weight (MW) of the polymer backbone is lkDa to 300kDa, such as 10 to 200 kDa or 20 to 100 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is 25 to 85 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is 30 to 60 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is 40 to 60 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is 40 to 50 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is about 40 kDa. In some embodiments, the molecular weight (MW) of the polymer backbone is about 50 kDa. It is understood that the MW of the polymer backbone refers to the MW of the polymer without the glycan groups present, e.g., prior to conjugation of the glycan groups of formula (V).
[0097] Preferably, the glycan groups that are attached to the polymer backbone are identical compounds of formula (XI), or include two or more different compounds formula (XI). Typically, the polymer further comprises spacer moieties attached to side chain groups of the repeat units of the polymer to provide for linking to the glycan groups. In some embodiments, the polymer is an amino acid polymer, including lysine residues to which spacer moieties are attached, which spacer moieties themselves include reactive moieties (e.g., chemoselective ligation groups) that link to a compatible group of a gly can-linker compound (e.g., of formula (V), as described herein). Preferred examples are described herein.
[0098] In some embodiments, the percentage of loading of the glycan group onto the polymer backbone is between 10 and 90%, preferably between 20 and 70%, between 20 and 60% or between 20 and 50%. The loading refers to the % of repeat units of the polymer backbone that are linked to a glycan group (e.g., as described herein). When the polymer is an amino acid polymer, the loading refers to the % of amino acid residues of the polypeptide that are linked to a glycan group (e.g., as described herein). In some embodiments, the loading of the amino acid polymer is determined synthetically by controlling the equivalents of glycan-linker compounds of formula (V) that are coupled to the polymer backbone. In some embodiments, the loading of the amino acid polymer is determined by controlling the composition of the polypeptide backbone and limiting the number of residues that are capable of coupling with glycan-linker compounds of formula (V).
[0099] The percentage of loading of the glycan group onto the polymer backbone is typically and preferably determined by NMR spectroscopy and refers to % mole/mole. In some embodiments, the loading of a polymeric compound of this disclosure is determined using a quantitative NMR method based on a relative determination of integrals of interest. In some embodiments, the loading of a polymeric compound of this disclosure is determined using a quantitative NMR method based on an absolute concentration determination by comparing integrals of interest to standard. [0100] Further particular examples of polymers of this disclosure are described in WO 2017046172, the disclosure of which is incorporated herein by reference in its entirety.
[0101] When the polymer is an amino acid polymer (also referred to as polypeptide) some side chain residues of the polymer backbone that are not attached to glycan groups of this disclosure can be modified to incorporate a water solubilizing substituent. For example, when the amino acid polymer includes lysine residues, some of the lysine residues may be capped with a water solubilizing substituent via amide bond formation, e.g., to an active ester group of a water solubilizing group precursor. In some embodiments, when the polymer is poly-lysine (e.g., as described herein) and a fraction of the lysine side chains are loaded with glycan groups, then substantially all of the remaining (uncapped) lysine side chains in the poly-lysine polymer are capped with a water solubilizing substituent.
[0102] The terms “water solubilizing substituent” and “water soluble group” are used interchangeably and refer to a moiety, group or molecule that is hydrophilic and imparts improved water solubility upon a compound to which it is attached. The water solubilizing substituent can be charged under physiological conditions, or can be a neutral hydrophilic group.
[0103] In some embodiments, the water solubilizing substituent is a polyol group. In some embodiments, the water solubilizing substituent includes a (Ci-Cg)-alkyl substituted with one, two or more hydroxyl groups. In some embodiments, the water solubilizing substituent includes a dihydroxypropyl or dihydroxyethyl group. A variety of chemoselective ligation groups and other linking groups or spacers can be utilized to link the water solubilizing substituent to the polymer. In some embodiments, the water solubilizing substituent is a group that can be charged under suitable aqueous conditions, e.g., a positively charged or negatively charged group, or a salt thereof, under physiological conditions. In some embodiments, the water solubilizing substituent is a carboxylic acid or a salt thereof. In some embodiments, the water solubilizing substituent is a sulfonic acid or a salt thereof.
[0104] In some embodiments, the water solubilizing substituent is 2,3-dihydroxypropylthioacetyl.
5.1.5.2 Supports
[0105] In some embodiments, the moiety of interest Y is a support. A variety of supports may be utilized. In general terms, a support is composed of an inert material that is, or can be, functionalized to provide for attachment of a linked compound or moiety. In some embodiments, the support can be referred to as a solid support. Supports of interest include, but are not limited to, beads, nanoparticles, planar supports, slides, microtiter plates (e.g., 96-well or other sized plates), membranes, monoliths, and the like. In some embodiments, the support is a bead. In some embodiments, utilization of monolithic solid-supports, membranes or planar solid supports, can be advantageous. In some embodiments, the support, e.g., beads, is biocompatible and suitable for use in an ex vivo application. [0106] The support can be composed of a variety of materials, including but not limited to, agarose, Sepharose, polystyrene, divinylbenzene, silica, silica-based material, metal-based material, silicone, nitrocellulose, polymethacrylate, polyacrylate, and the like. Any immobilization chemistry can be selected as desired depending on the solid-support material.
[0107] In some embodiments, the support is a bead suitable for ex vivo applications, e.g., where a bead immobilized-glycan can be used extracorporeally as part of an immune -adsorption support or apparatus configured to remove target autoantibodies from a biological sample. Beads of interest include, but are not limited to, an agarose, a sepharose, a dextran, a cellulose, chitin, chitosan, derivatives thereof, an organic or inorganic porous material, a magnetic bead, or a micro bead.
[0108] In some embodiments, the support is a bead, such as agarose beads or Sepharose beads (e.g., N-hydroxysuccinimide (NHS) activated Sepharose beads, or epoxy-activated Sepharose beads). In some embodiments, the beads are composed of paramagnetic core (e.g. Dynabeads).
[0109] The glycan-support, e.g., gly can-bead, conjugates can be incorporated into an external immune-adsorption apparatus suitable for use in removing target autoantibodies from a biological sample, e.g., a blood, serum or plasma sample of a patient. The beads can be utilized in a column or cartridge format, e.g., a format suitable for achieving separation of target anti-GMl autoantibodies from a biological sample. In some embodiments, the beads are suitable for chromatography.
5.1.5.3 Carrier Molecules
[0110] In some embodiments, the moiety of interest Y is a carrier molecule. The terms “carrier” and “carrier molecule” can refer to a biomolecule having multiple attachment points to which a glycan group can be linked. The biomolecule can be one that is capable of transport of a linked compound through a biological system. In some embodiments, Y is a biomolecule. In some embodiments, Y is a biomolecule selected from protein, polynucleotide, polysaccharide, peptide, glycoprotein, lipid, enzyme, antibody, and antibody fragment.
[0111] In some embodiments of formula (I)-(II), Y is a carrier molecule, and m is 2 to 50, such as 2 to 40, 2 to 30, 2 to 20 or 2 to 10.
[0112] A glycan-linker compound of formula (V) having a chemoselective ligation group can be conjugated to compatible amino acid side chain groups of a carrier protein. In some embodiments, the protein is itself modified to incorporate a chemoselective ligation group suitable for conjugation to a compatible glycan-linker compound of formula (V). In some embodiments, Z2 is an amine-reactive group (e.g., an active ester, e.g., NHS ester) and can be used to conjugate one, two or more glycan groups to the protein, e.g., via conjugation to lysine sidechain residues of the protein. In some embodiments, Z2 is a cysteine-reactive group (e.g., a maleimide) and can be used to conjugate one, two or more glycan groups to the protein. In some embodiments, Z2 is a thiol (-SH) and can be used to conjugate one, two or more glycan groups to the protein, where the protein has been modified to incorporate a thiol-reactive group, such as a maleimide. The ratio of glycan groups to carrier protein depends on the conjugation chemistry and stoichiometry of the ligation reaction. In some embodiments of formula (I)-(II), m is an average number of glycan groups per protein, such as m is 2 to 50 glycan groups per protein, such as 2 to 40, 2 to 30, 2 to 20 or 2 to 10 glycan groups per protein. [0113] In some embodiments of formula (I)-(II), where Y is a carrier molecule, the conjugate itself can be immobilized on a support, e.g., via non-covalent absorption of the carrier protein to a support surface. Such immobilized conjugates can find use in diagnostic applications.
5.1.6. Anti-GMl Antibody-Binding Compounds and Conjugates
[0114] As summarized above, several glycan groups can be linked to a moiety of interest (e.g., as described herein) to provide an anti-GMl antibody-binding compound or conjugate capable of multivalent binding to the antibody.
[0115] In some embodiments, the anti-GMl antibody-binding compound or conjugate is of formula (I) comprising one or more glycans linked to a moiety of interest:
Figure imgf000025_0001
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group;
Z1 is -0-, -S-, -NR2- or -C(R2)2-, wherein each R2 is independently selected from H, (C1-C4)- alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5;
Ar is optionally substituted aryl or optionally substituted heteroaryl (e.g., as described herein);
L1 is a linker (e.g., as described herein); m is one or more; and
Y is a moiety of interest (e.g., as described herein).
[0116] In some embodiments, m is 2 or more, such as 3 or more, 4 or more, 5 or more, 10 or more,
20 or more, 30 or more, 40 or more, 50 or more, 100 or more. The moiety of interest Y can have multiple attachment points for conjugation to two or more or several gly can-linker groups (e.g., as described herein). [0117] In some embodiments, Y is a polymer (e.g., as described herein), and m depends on the length of the polymer and the % loading (i.e., loading based on a molar ratio). In some embodiments, m is 2 to 1000, such as 20 to 1000, 50 to 500, 50 to 400, 60 to 300, 60 to 200, 80 to 150 or 80 to 120.
[0118] In some embodiments, Y is a support (e.g., as described herein), and m depends on the mass loading of glycan groups on the support. In such cases, the conjugate material can be characterized in terms of a loading in terms of mass ratio of glycan groups to the underlying support material. Alternatively, the loading can be characterized in terms of the density of functional attachment points on the support.
[0119] In some embodiments, the anti-GMl antibody-binding polymeric compound of formula (I) is of formula (II):
Figure imgf000026_0001
wherein: q is 0 to 4; and each R11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
[0120] In some embodiments of formula (I)-(II), the glycan group is as defined in any one of the embodiments of the glycan groups of formula (XI)-(XII) described here.
[0121] In some embodiments of formula (I)-(II), the compound is selected from formula (Ila)-(IIe):
Figure imgf000026_0002
(Ha), PCT/IB2022/000224
Figure imgf000028_0001
(He), or a salt thereof, wherein:
-L1- is -Z11-(Ci-C6)-alkyl-Z12-(Ci-C6)-alkyl-S-CH2CO-;
Z11 is absent, CO, C(=0)NH, SO2NH, O, S, NH, -NHC(=0)-, -NHC(=0)NH-, or - NHC(=S)NH-;
Z12 is absent, -NHC(=0)-, C(=0)NH, -NHC(=NH)-, SO2NH, O, S, or NH; and Y is a moiety of interest (e.g., as described herein). In some embodiments of these compounds, -L1- comprises -(Ci-C6)-alkyl-amido-(Ci-C6)-alkyl-S-CH2CO-, and connects via an amide bond to an amine group of Y. In some embodiments of these compounds, -L1- comprises - (CH2)2NHCO(CH2)3-S-CH2CO-, and connects via an amide bond to an amine group of Y.
[0122] In some embodiments of formula (I)-(IIe), the moiety of interest Y is a polymer.
[0123] In some embodiments of formula (I)-(IIe), Y is an amino acid polymer (e.g., polylysine). In some embodiments of formula (I)-(IIe), Y is an amino acid polymer backbone having a mean length of 10 to 5000 amino acid residues, such as 20 to 2000, 30 to 1000, or 50 to 800, 50 to 500, 100 to 500, 200 to 500, or 300 to 400 amino acid residues.
[0124] In some embodiments of formula (I)-(IIe), Y is an amino acid polymer backbone having a mean length of 360-440 amino acid residues, such as 380-420 amino acid residues, or 390-410 amino acid residues, e.g., about 400 amino acid residues.
[0125] In some embodiments of formula (I)-(IIe), the polymer is an alpha-amino acid polymer, and the polymer is composed of amino acid resides selected from lysine, ornithine, glutamic acid, aspartic acid, and serine.
[0126] In some embodiments of formula (I)-(IIe), the polymer is poly-lysine. In some embodiments of formula (I)-(IIe), the polymer is poly- -lysine. In some embodiments of formula (I)-(IIe), the polymer is poly-D-lysine. In some embodiments of formula (I)-(IIe), the polymer is a co-polymer of lysine residues and one or more additional amino acid residues (e.g., as described herein). In some embodiments of formula (I)-(IIe), the polymer is poly-aspartic acid or poly-glutamic acid.
[0127] In some embodiments of formula (I)-(IIe), the polymer is an alpha-amino acid polymer, and the percentage of loading of the glycan group onto the polymer backbone is 15 to 50%, such as 20 to 40%, or 25 to 40%. In some embodiments of formula (I)-(IIe), the loading of the glycan group onto the polymer backbone is 25 to 30%. In some embodiments of formula (I)-(IIe), the loading of the glycan group onto the polymer backbone is 30 to 40%, such as 30 to 35%, or 36 to 40%. In some embodiments of formula (I)-(IIe), the loading of the glycan group onto the polymer backbone is about 36%. In some embodiments of formula (I)-(IIe), loading of the glycan group onto the polymer backbone is about 29%. In preferred embodiments, the polymer backbone is poly-lysine, such as poly- -lysine or poly-D-lysine.
[0128] In some embodiments of formula (I)-(IIe), the polymer is poly-lysine and further comprises spacer moieties attached to the amine side chain groups of the lysine residues to provide for linking to the glycan groups (e.g., of formula (V)). In some embodiments, the lysine residues of the polymer backbone are capped with spacer moieties (e.g., halo-acetyl groups, such as C1-CH2-C(=0)-) that themselves include a thiol-reactive moiety suitable for conjugation with a thiol containing glycan- ligand compound of formula (V) (e.g., as described herein).
[0129] Thus, in some embodiments of formula (I)-(IIe), L1 is -(CH2)2NH(C=0)(CH2)3S-CH2-(C=0)- connecting the phenyl ring of the glycan mimic to an amine -containing group, e.g., a lysine side chain residue of a polypeptide backbone.
[0130] A variety of spacer groups can be used to provide for linkage between alternative amino acid residues (such as aspartic acid or glutamic acid residues), and glycan-linker compounds of formula (V). In some embodiments, a glycan-linker compounds of formula (V) is conjugated directly to an amino acid side chain group with an intermediate spacer.
[0131] In some embodiments of formula (I)-(IIe), the polymer is poly-lysine polymer where a fraction of the lysine side chains are loaded with glycan groups (e.g., as described above) and substantially all of the remaining lysine side chains in the poly-lysine polymer are capped with a water solubilizing substituent. In some embodiments, the water solubilizing substituent attached to the lysine side chains is 2,3-dihydroxypropylthioacetyl-.
[0132] Exemplary polymeric compounds of formula (I)-(IIe), are described in the experimental section.
5.1.7. Multimeric Compounds and Conjugates
[0133] Also provided are multimeric anti-GMl antibody-binding compounds or conjugates, where multiple (e.g., two, three or more) glycan-containing compounds or conjugates are linked together, e.g., via linkage to their moieties of interest (Y). Multimerization can be achieved via use of a branching moiety having two, three or more linked functional groups that provide points of attachment to which a suitable functional group of the moiety of interest (Y) can be conjugated. Any suitable chemoselective ligation group chemistry, e.g., as described herein can be used to connect a branching moiety to each monomeric anti-GMl antibody-binding compounds or conjugate. In some embodiments, the moiety of interest Y is a polymer. In some embodiments, the moiety of interest Y is a polypeptide or amino acid polymer. [0134] Accordingly, in some embodiments, a multimeric anti-GMl antibody-binding compound is of formula (IV):
Figure imgf000030_0001
or a pharmaceutically acceptable salt thereof, wherein:
M1, M2 and M3 are each independently monomeric units that together provide a polymer backbone (P) (e.g., as described herein) having a mean length of 10-5000 amino acid residues, x is 10 to 50 mol% of M1 units in the polymer backbone (P); y is 0 to 90 mol% of M2 units in the polymer backbone (P);
Y2 is an optional terminal group;
L1 is a linker that connects a glycan (G) to the M1 monomeric unit;
WSG is a water solubilizing group linked to the M2 monomeric unit;
L2 is an optional linker connecting the polymer backbone (P) to B;
B is a branching moiety that covalently links to “n” polymer backbones, wherein n is 2 to 6; and each G is independently a glycan group that mimics GM1.
[0135] In some embodiments of formula (IV), the G groups linked to the polymer backbone (P) include at least one glycan group according to any one of the embodiments of formula (XI) -(XII) as described herein.
[0136] In some embodiments of formula (IV), each G is independently of formula (XI) or (XIV)
Figure imgf000030_0002
or
Figure imgf000031_0001
wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group.
Z1 and Z3 are selected from -0-, -S-, -NR2- and -C(R2)2-, wherein each R2 is independently selected from H, (Ci-C -alkyl, (Ci-C- -alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and - OCH2CH2C6H5; and
Ar is selected from optionally substituted aryl, and optionally substituted heteroaryl.
[0137] In some embodiments of formula (IV), the G groups linked to the polymer backbone (P) include at least one glycan group of formula (XIV). In some embodiments of formula (XIV), R1 is a sialic acid group, e.g., of formula (XHIa) as described herein.
[0138] . In some embodiments of formula (XIV), R1 is optionally substituted carboxymethyl group, e.g., of formula (XHIb) as described herein.
[0139] In some embodiments of formula (IV), M1, M2 and M3 are each independently monomeric units of a polymer selected from amino acid polymers, polyacrylate polymers or copolymers, poly(meth)acrylate polymers or copolymers, N-vinyl-2-pyrrolidone-vinylalcohol copolymers, chitosan polymers, and polyphosphazene polymers.
[0140] In some embodiments of formula (IV), M1, M2 and M3 are each independently monomeric units that together provide a polymer backbone (P) having a mean length of 20 to 2000 repeat units, such as 30 to 1000, 50 to 800, 50 to 300, 50 to 200, or 100 to 200 repeat units.
[0141] The M1 monomer units can include a sidechain functional group capable of conjugation to a linked glycan group (e.g., as described herein). The M2 monomer units can include a sidechain functional group capable of conjugation to a WSG (e.g., as described herein). Alternatively, the M2 monomer units selected for incorporation into the polymer backbone already have a hydrophilic or charged WSG sidechain group as part of the repeat unit. Any convenient M3 monomer units can be selected to impart a desirable property upon the polymer backbone. In general terms the M1, M2 and M3 monomer units are all of the same monomer class and monomer chemistry. In some embodiments, the M1, M2 and/or M3 monomer units are assembled via a random polymerization by combining desired ratios of the monomer units for polymerization. In some embodiments, the M1, M2 and/or M3 monomer units are assembled via a block copolymer polymerization method. In some embodiments, the M1, M2 and/or M3 monomer units are assembled in a stepwise fashion and have a defined length and sequence. [0142] In some embodiments of formula (IV), the multimeric compound includes ‘m’ polymer backbones (P) that together have a total number of repeat units of 150 to 1000, such as 150 to 8000, 150 to 600, or 300 to 600 repeat units.
[0143] In some embodiments of formula (IV), M1, M2 and M3 are each independently amino acid residues that together provide an amino acid polymer backbone (P) having a mean length of 20 to 2000 amino acid residues, such as 30 to 1000, 50 to 800, 50 to 300, 50 to 200, or 100 to 200 amino acid residues.
[0144] In some embodiments of formula (IV), the multimeric compound includes ‘m’ amino acid polymer backbones (P) that together have a total number of amino acid residues of 150 to 1000, such as 150 to 8000, 150 to 600, or 300 to 600 amino acid residues.
[0145] In some embodiments of formula (IV), x is 15 to 50 mol% of M1 units in the polymer backbone (P), such as 20 to 40 mol% or 25 to 40 mol%, or 30 to 40 mol%.
[0146] In some embodiments of formula (IV), x + y is 1, such that M3 is absent.
[0147] In some embodiments of formula (IV), M3 is present, and in some cases, present at 1 to 25 mol%, such as 1 to 20 mol%, or 1 to 10 mol%.
[0148] In some embodiments of formula (IV), y is > 0 mol%. In some embodiments of formula (IV), y is at least 10 mol%, such as at least 20 mol%, at least 40 mol%, or at least 50 mol%.
[0149] In some embodiments of formula (IV), y is 50 to 75 mol%, such as 60 to 75 mol%.
[0150] In some embodiments of formula (IV), Y2 is an optional terminal group. It is understood that a terminal group can be any suitable group or moiety, such as H-, a terminal group of a monomer, a capping group, a protecting group, a linked glycan, a linked moiety of interest, or a residue of polymer synthesis, such as a polymerization initiator. It is understood that a linker (e.g., L2) can be connected to a terminal of the polymer, e.g., directly to a terminal monomer unit, or via an optional terminal group, such as a residue of a polymerization initiator (e.g., as described herein).
[0151] In some embodiments of formula (IV), the polymer P is a linear amino acid polymer, and Y2 is a terminal group located at the N-terminal amino acid residue. In some embodiments of formula (IV), the polymer P is a linear amino acid polymer, and Y2 is a terminal group located at the C- terminal amino acid residue.
[0152] In some embodiments of formula (IV), the polymer P is an amino acid polymer where each M1 is an amino acid that includes a sidechain group capable of conjugation (e.g., via chemoselective ligation) to a -L'-G is a glycan group of formula (XI)-(XII) (e.g., as described herein). In some embodiments of formula (IV), each M1 is selected from lysine, ornithine, aspartic acid, and glutamic acid, where the glycan group can be attached via amide bond formation. In some embodiments of formula (IV), each M1 is an amino acid residue having a sidechain group that has been modified with an intermediate linking group that install a desirable sidechain chemosselective ligation group compatible with a complementary group of the glycan-linker precursor. For example, a lysine monomer unit having a sidechain modified with a thiol-reactive group, e.g., a haloacetamide group (- COCH2CI).
[0153] Similarly, M2 can be any convenient amino acid unit that is capable of conjugation to a water solubilizing group. In some embodiments of formula (IV), each M2 is selected from lysine, ornithine, aspartic acid, and glutamic acid, where the water solubilizing group can be attached via amide bond formation. In some embodiments of formula (IV), each M2 is an amino acid residue having a sidechain group that has been modified with an intermediate linking group that install a desirable sidechain chemoselective ligation group compatible with a complementary group of the WSG precursor. For example, a lysine monomer unit having a sidechain modified with a thiol-reactive group, e.g., a haloacetamide group (-COCH2CI).
[0154] The M3 repeat unit can be any convenient amino acid. In some instances, the M3 can be present at a sufficient level (mol %) that together with the M1 and M2 repeat units provide a compound having desirable glycan loading and/or physical properties. In some embodiments, M3 is an amino acid residue selected from glycine, alanine, or serine.
[0155] In some embodiments of formula (IV), the polymer P is an amino acid polymer where each M1 is lysine residue, and each -L'-G is a glycan group of formula (XI)-(XII) (e.g., as described herein). In some embodiments of formula (IV), the polymer P is an amino acid polymer where each M2 is lysine residue linked to a water solubilizing group via a spacer (e.g., haloacetyl spacer conjugated with a thiol containing water soluble group (e.g., thioglycerol). In some embodiments M3 is absent and M1 and M2 are lysine residues.
[0156] It is understood that the multimeric anti-GMl antibody-binding compound of formula (IV) may be represented by an alternative formula, e.g., a formula as follows that depicts the several glycan groups linked to the polymer backbone, which is itself multimerized.
[0157] Accordingly, in some embodiments, the anti-GMl antibody-binding multimeric compound is of formula (III):
Figure imgf000033_0001
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group. Z1 is -0-, -S-, -NR2- or -C(R2)2-, wherein each R2 is independently selected from H, (C1-C4)- alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5;
Ar is optionally substituted aryl or optionally substituted heteroaryl (e.g., monocyclic aryl or heteroaryl, or bicyclic aryl or heteroaryl);
L1 is a linker; m is at least 2;
P is a polymer;
L2 is an optional linker; n is 2 to 6; and B is a branching moiety.
[0158] In some embodiments of formula (III)-(IV), n is 2 whereby the compound is dimeric. In some embodiments, the compound is homo-dimeric. In some embodiments, the compound is hetero- dimeric.
[0159] In some embodiments of formula (III)-(IV), n is 3 whereby the compound is trimeric.
[0160] Higher valency multimers can also be provided, e.g., by configuration of multiple branching moieties that produce a dendrimer.
[0161] The branching moiety B can be a distinct and different moiety from the polymer P, e.g., B is a group or molecule that is different from any repeat unit of the polymer P, and can be attached to the polymer P via an orthogonal chemistry as compared to the chemoselective ligation chemistry used to achieve attachment of the gly can-linker compounds to the polymer backbone.
[0162] In some embodiments of formula (III)-(IV), B is aryl or heteroaryl group substituted at two or more positions with L2 linkers, and optionally further substituted. In some embodiments of formula (III)-(IV), B is derived from an aryl or heteroaryl group substituted at two or more positions with carboxylic acid groups, or derivatives thereof, to which optional linkers (L2) can be attached, and optionally further substituted.
[0163] In some embodiments of formula (III)-(IV), B is optionally substituted phenyl substituted at two or more positions via linking moieties to L2 linkers, where the L2 linkers can include chemoselective ligation groups for attachment to P polymers.
[0164] In some embodiments of formula (IV)-(III), B is a trisubstituted aryl or heteroaryl group, and L2 is -CONH-(Ci-Cg)-alkyl-NH-. In some embodiments of formula (IV)-(III), B is a trisubstituted phenyl group, and L2 is -CONH-CH2CH2-NH-. In some embodiments of formula (IV)-(III), P is an amino acid polymer (e.g., poly-lysine) and L2 connects to the C-terminal of the polymer via an amide bond. [0165] In some embodiments of formula (III)-(IV), B is
Figure imgf000035_0001
where each carbonyl group links to an L2 linker of formula (IV)-(III), e.g., via an amide linkage. [0166] In some embodiments of formula (III), each P is an amino acid polymer backbone (e.g., as described herein), and each L2 links the C-terminal residue of the amino acid polymer backbone to a carbonyl or carboxyl group of B via amide bonds. In some embodiments, the amino acid polymer backbone is linear.
[0167] In some embodiments of formula (III), each P is an amino acid polymer backbone, and each L2 links the N-terminal residue of the a-amino acid polymer backbone to B. In some embodiments, the amino acid polymer backbone is linear.
[0168] Any suitable linkers (e.g., as described herein) can be incorporated into the compounds of formula (III) to link the branching moiety B with the two, three or more polymers P.
[0169] In some embodiments of formula (III), each L2 is a linear linker comprising one or more linking moieties independently selected from -Ci-6-alkylene-, -NHCO-Ci-6-alkylene-, -CONH-CI-6- alkylene-, -0(CH2)p-, -(OCH2CH2)p-, -NHCO-, -CONH-, -NHS02-, -S02NH-, -CO-, -S02-
0-, — S — , pyrrolidine-2, 5-dione, 1,2,3-triazolyl, -NH-, and -NMe-, wherein p is 1 to 10.
[0170] In some embodiments of formula (III), each L2 is -NH-(CH2)P-NH-, wherein p is 2 to 6. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4.
[0171] In some embodiments of formula (III), Ar is
Figure imgf000035_0002
wherein: q is 0 to 4; and each R11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
[0172] In some embodiments of formula (III)-(IV), the compound includes a multitude of glycan- linker groups of formula (XI)-(XII), as defined in any one of the embodiments described herein. [0173] Exemplary polymeric compounds of formula (I)-(IIe), are described in the experimental section. 5.1.8. Pharmaceutical Compositions
[0174] Also provided by this disclosure are pharmaceutical compositions comprising a compound (e.g., as described herein). In some embodiments, the pharmaceutical composition includes a polymeric compound of formula (I)-(IV), e.g., as described by any of the embodiments set forth herein.
[0175] Pharmaceutical compositions for parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular administration, to warm-blooded animals, especially humans, are considered. The compositions comprise the active ingredient(s) alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the active ingredient(s) depends upon the age, weight, and individual condition of the patient, the individual pharmacokinetic data, and the mode of administration.
[0176] For parenteral administration preference is given to the use of suspensions or dispersions of the carbohydrate polymer of this disclosure, especially isotonic aqueous dispersions or suspensions which, for example, can be made up shortly before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, viscosity-increasing agents, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.
[0177] Suitable carriers for enteral administration, such as nasal, buccal, rectal or oral administration, are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example com, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
[0178] Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinyl-pyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl-methylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient(s). [0179] Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as com starch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.
[0180] The mentioned pharmaceutical compositions according to this disclosure may contain separate tablets, granules or other forms of orally acceptable formulation of the active ingredients, or may contain a mixture of active ingredients in one suitable pharmaceutical dosage form, as described above. In particular the separate orally acceptable formulations or the mixture in one suitable pharmaceutical dosage form may be slow release and controlled release pharmaceutical compositions. [0181] The pharmaceutical compositions can comprise from approximately 0.1% to approximately 95% active ingredient or mixture of active ingredients. In some embodiments, the composition is in a single-dose administration form.
[0182] This disclosure also relates to the mentioned pharmaceutical compositions as medicaments in the treatment of neurological diseases associated with anti-glycan antibodies, particularly immune- mediated neuropathies.
5.2. Methods
[0183] Another aspect of this disclosure relates to a method of inhibiting or specifically binding an anti-GMl antibody in a sample. In some embodiments, the method includes contacting a sample comprising the anti-GMl antibody with an effective amount of a compound or conjugate of this disclosure (e.g., of formula (I)-(IV) as described herein). In some embodiments, the compound or conjugate is a polymeric compound of formula (I) where Y is a polymer, such as an amino acid polymer (e.g., as described herein). In some embodiments, the compound or conjugate is a conjugate of formula (I) where Y is a support.
[0184] In some embodiments, the sample is a biological sample. The biological sample can be obtained from a subject and can be any suitable body fluid sample. Body fluids that are useful for binding of anti-GMl antibodies include, but are not limited to, serum, plasma, whole blood, cerebrospinal fluid, and extracts from solid tissue. In some embodiments, the biological sample is serum or plasma.
[0185] Another aspect of this disclosure relates to a method of treating particular anti-GMl antibody mediated neuropathy. In some embodiments, the anti-GMl antibody mediated neuropathy is Guillain-Barre-Syndrome (GBS), including variants of GBS such as acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) or a pharyngeal-cervical-brachial variant of GBS, or the chronic multifocal motor neuropathy (MMN).
[0186] The present invention relates furthermore to a method of treatment of neurological diseases associated with anti-glycan antibodies, particularly immune-mediated neuropathies, which comprises administering a composition according to this disclosure in a quantity effective against said disease, to a warm-blooded animal requiring such treatment. The pharmaceutical compositions can be administered prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily, weekly or monthly dose administered is from approximately 0.01 g to approximately 5 g, preferably from approximately 0.1 g to approximately 1.5 g, of the active ingredients in a composition of the present invention.
[0187] This disclosure includes methods of treatment of neurological diseases associated with anti- glycan antibodies, particularly immune-mediated neuropathies. The methods can include extracorporeal separation of target anti-GMl autoantibodies from a biological sample, e.g. a biological fluid such as peripheral blood (or other biological fluids such as whole blood, arterial blood, cerebrospinal fluid, peritoneal or pleural fluid, etc.) of a warm-blooded animal (for example a human patient) requiring such treatment. The extracorporeal separation of target anti-GMl autoantibodies from a biological sample of a patient can be achieved by contacting the sample with an effective amount of a compound of this disclosure that is immobilized on a support in a format suitable for achieving separation. In some embodiments, the support is beads. In certain embodiments, the beads are packed in a column or cartridge, and the biological sample is flowed through the column or cartridge to remove the target anti-GMl autoantibodies from the biological sample. In some embodiments, the beads are magnetic and subsequent application of a strong magnet will pull down the beads along with the target anti-GMl autoantibodies, allowing those antibodies to be removed from the biological sample. When the biological sample, e.g., peripheral blood, has been treated with the immobilized compound of this disclosure, it can be returned to the subject or patient. In some embodiments, the beads are biocompatible and suitable for use in an ex vivo application. The immobilized compositions (e.g., beads) can be utilized prophylactically or therapeutically.
5.3. Diagnostics
[0188] Another aspect of this disclosure relates to a method of diagnosis of neurological diseases, particularly immune-mediated neuropathies, wherein the level of antibodies (e.g. IgM/IgG) against glycans of the nervous system, particularly GM1 ganglioside, is determined in a biological sample. A high level of anti-GMl autoantibodies in the sample is indicative of the development and the severity of a particular anti-GMl antibody mediated neuropathy. In some embodiments, the anti-GMl antibody mediated neuropathy is Guillain-Barre-Syndrome (GBS), including variants of GBS such as acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) or a pharyngeal-cervical-brachial variant of GBS, or the chronic multifocal motor neuropathy (MMN).
[0189] The biological sample is obtained from the patient and can be any suitable body fluid sample. Body fluids that are useful for determination of antibodies against GM1 glycoepitopes include, but are not limited to, serum, plasma, whole blood, cerebrospinal fluid, and extracts from solid tissue. In some embodiments, the biological sample is serum.
[0190] Any suitable method may be used for the determination of the level of anti-GMl autoantibodies in the biological sample. Methods considered are, e.g., ELISA, RIA, EIA, Flow Cytometry, electrochemiluminescence system or microarray analysis.
[0191] In some embodiments, the method used for the determination of anti-GMl autoantibodies in the biological sample, such as a human body fluid, e.g. in serum, involves capture of the autoantibodies with beads loaded with compounds of this disclosure. In some embodiments, the beads utilized in the diagnostic assay include gly can-linker compounds of formula (V). The beads can be adapted for use in a variety of assays to qualitatively or quantitatively assess the presence or level of anti-GMl autoantibodies in the sample. In some instances, anti-GMl autoantibodies bound to the glycan-linked beads can be detected using a secondary antibody labelled with a dye. In some cases an ELISA assay is performed in the bead format to detect bead bound anti-GMl autoantibodies. In some embodiments, a flow cytometry assay is performed in the bead format to detect bead bound anti-GMl autoantibodies. Detection can be achieved using a variety of methods, including flow cytometry, or a bead based sandwich assay with fluorescence microscopy.
[0192] In some embodiments, the method used for the determination of anti-GMl autoantibodies in human body fluids, e.g. in serum, is an ELISA. In such an embodiment, microtiter plates are coated with glycan-linker compounds of formula (V) or preferably conjugates of this disclosure (e.g., peptidic compounds, or carrier molecule conjugates, or the like) comprising such compounds of formula (V) as substituents. The plates are then blocked and the biological sample or a standard solution is loaded. After incubation, an anti-IgM/IgG antibody is applied, e.g. an anti-IgM or anti-IgG antibody directly conjugated with a suitable label, e.g. with an enzyme for chromogenic detection. Alternatively, a polyclonal rabbit (or mouse) anti-IgM / anti-IgG antibody is added. A second antibody detecting the particular type of the anti-IgM / anti-IgG antibody, e.g. an anti-rabbit (or anti mouse) antibody, conjugated with a suitable label, e.g. the enzyme for chromogenic detection as above, is then added. Finally, the plate is developed with a substrate for the label in order to detect and quantify the label, being a measure for the presence and amount of anti-GMl autoantibodies. If the label is an enzyme for chromogenic detection, the substrate is a colour-generating substrate of the conjugated enzyme. The colour reaction is then detected in a microplate reader and compared to standards.
[0193] It is also possible to use antibody fragments. Suitable labels are chromogenic labels, i.e. enzymes which can be used to convert a substrate to a detectable colored or fluorescent compound, spectroscopic labels, e.g. fluorescent labels or labels presenting a visible color, affinity labels which may be developed by a further compound specific for the label and allowing easy detection and quantification, or any other label used in standard ELISA.
[0194] Other preferred methods of detection of target antibodies are radioimmunoassay or competitive immunoassay and chemiluminescence or electrochemiluminescence detection on automated commercial analytical robots. Microparticle enhanced fluorescence, fluorescence polarized methodologies, or mass spectrometry may also be used. Detection devices, e.g. microarrays, are useful components as readout systems for target antibodies.
[0195] In a further aspect, the present disclosure relates to a kit suitable for an assay as described above, in particular an ELISA, comprising compounds of formula (IV) or conjugates comprising such compounds as substituents (e.g., as described herein). The kits can further contain anti-IgM / anti-IgG antibodies (or anti-IgM/IgG antibody fragments) carrying a suitable label, or anti-IgM / anti-IgG antibodies and second antibodies carrying such a suitable label, and/or reagents or equipment to detect the label, e.g. reagents reacting with enzymes used as labels and indicating the presence of such a label by a color formation or fluorescence, standard equipment, such as microtiter plates, pipettes and the like, standard solutions and wash solutions.
[0196] The ELISA can be also designed in a way that patient blood or serum samples are used for the coating of microtiter plates with the subsequent detection of anti-GMl antibodies with labelled compounds of formula (V) (e.g., a compound linked to a detectable label) or conjugates or polymeric compounds comprising such compounds as substituents, and a detectable label. The label is either directly detectable or indirectly detectable, e.g., via an antibody or other binding protein. In some instances, the detectable label is a fluorophore or chromophore dye.
5.4. Definitions
[0197] The term “gly coepitope” refers to the carbohydrate moiety or glycan group that is recognized by an anti-GMl antibody.
[0198] The term “reducing end”, as used herein in the context of the glycoepitope of the present invention and of the specific inventive compounds, refers to the monosaccharide of the glycoepitope with a free anomeric carbon that is not involved in a glycosidic bond, wherein said free anomeric carbon bears a hemiacetal group.
[0199] The term “(Ci-Cn)-alkyl” refers to straight or branched carbon chain of 1 to n carbon atoms. The term “(Ci-C4)-alkyl” includes butyl, such as /7-butyl sec-butyl, Ao-butyl, tert- butyl, propyl, such as «-propyl or iso- propyl, ethyl or methyl. In some embodiments, the term “(Ci-C4)-alkyl” refers to methyl or ethyl, «-propyl or 750-propyl. In some embodiments, the term “(Ci-C4)-alkyl” refers to methyl. Correspondingly, the term CVCValkyl” refers to straight or branched chain of 1 to 6 carbon atoms.
[0200] The term “(Ci-Cn)-alkylene“ refers to a straight or branched bivalent alkyl chain of 1 to n carbon atoms, and includes, for example, -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(CH3)- CH2-, or -CH(CH2CH3)-, etc.
[0201] The term “(Ci-Cn)-alkoxy” refers to an alkoxy with a straight or branched chain of 1 to n carbon atoms. The term “Ci-C4-alkoxy”, as used herein, refers to an alkoxy with a straight or branched chain of 1 to 4 carbon atoms and includes methoxy, ethoxy, propoxy, /.vo-propoxy. «- butoxy, vt'c-butoxy and fert-butoxy. Preferably, the term “Ci-C4-alkoxy”, as used herein, refers to methoxy, ethoxy, propoxy. Further preferably, the term “Ci-C4-alkoxy”, as used herein, refers to methoxy.
[0202] Double bonds in principle can have E- or Z-configuration. The compounds of this invention may therefore exist as isomeric mixtures or single isomers. If not specified both isomeric forms are intended.
[0203] Any asymmetric carbon atoms may be present in the ( R )-, (.V)- or (R, <S) -configuration, preferably in the ( R )- or (^-configuration. The compounds may thus be present as mixtures of isomers or as pure isomers, preferably as enantiomer-pure diastereomers.
[0204] The term “alkynyl” refers to is a straight or branched carbon chain or cyclic group comprising one or more, preferably one triple bond. Preferred are Ci-C4-alkynyl, such as propargyl or acetylenyl. An exemplary cyclic alkynyl group is a cyclooctyne, or substituted cyclooctyne.
[0205] The term "aryl” refers to a mono- or bicyclic system of aromatic rings with 5 to 12 carbon atoms optionally carrying substituents, such as phenyl, 1 -naphthyl or 2-naphthyl, or also a partially saturated bicyclic fused ring comprising a phenyl group, such as indanyl, indolinyl, dihydro- or tetrahydronaphthyl, all optionally substituted. Preferably, aryl is phenyl, indanyl, indolinyl or tetrahydronaphthyl, in particular phenyl.
[0206] The term "heteroaryl" refers to an aromatic mono- or bicyclic ring system containing at least one heteroatom, and preferably up to three heteroatoms selected from nitrogen, oxygen and sulfur as ring members. Heteroaryl rings do not contain adjacent oxygen atoms, adjacent sulfur atoms, or adjacent oxygen and sulfur atoms within the ring. Monocyclic heteroaryl preferably refers to 5 or 6 membered heteroaryl groups and bicyclic heteroaryl preferably refers to 9 or 10 membered fused-ring heteroaryl groups. Examples of heteroaryl include pyrrolyl, thienyl, fiiryl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, and benzo or pyridazo fused derivatives of such monocyclic heteroaryl groups, such as indolyl, benzimidazolyl, benzofuryl, quinolinyl, isoquinolinyl, quinazolinyl, pyrrolopyridine, imidazopyridine, or purinyl, all optionally substituted.
[0207] Preferably, the term "heteroaryl" refers to a 5- or 6-membered aromatic monocyclic ring system containing at least one heteroatom, and preferably up to three heteroatoms selected from nitrogen, oxygen and sulfur as ring members. Preferably, heteroaryl is pyridyl, pyrimdinyl, pyrazinyl, pyridazinyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, oxadiazolyl, triazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyrrolyl, indolyl, pyrrolopyridine or imidazopyridine; in particular pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, imidazolyl, thiazolyl, oxadiazolyl, triazolyl, indolyl, pyrrolopyridine or imidazopyridine
[0208] The term “optionally substituted aryl” refers to aryl substituted by up to five substituents, preferably up to two substituents. In optionally substituted aryl, preferably in optionally substituted phenyl, substituents are preferably and independently selected from Ci-Ct-alkyl, Ci-Ct-alkoxy, amino-Ci-Czt-alkyl, acylamino-Ci-Ct-alkyl, aryl-Ci-Ct-alkyl hydroxy, carboxy, cyano, C1-C4- alkoxycarbonyl, aminocarbonyl, hydroxylaminocarbonyl, tetrazolyl, hydroxysulfonyl, aminosulfonyl, hydroxy, halo, or nitro, in particular Ci-Ct-alkyl, Ci-Ct-alkoxy, amino-Ci-Ct-alkyl, acylamino-Ci-Ct- alkyl, carboxy, Ci-Ct-alkoxycarbonyl, aminocarbonyl, hydroxylaminocarbonyl, tetrazolyl, and aminosulfonyl.
[0209] The term “optionally substituted heteroaryl” refers to heteroaryl substituted by up to three substituents, preferably up to two substituents. In optionally substituted heteroaryl, substituents are preferably and independently selected from Ci-Ct-alkyl, Ci-Ct-alkoxy, halo-Ci-Ct-alkyl, hydroxy, Ci- Ct-alkoxycarbonyl, aminocarbonyl, hydroxylaminocarbonyl, tetrazolyl, aminosulfonyl, halo, aryl-Ci- Ct-alkyl, or nitro.
[0210] Cycloalkyl has preferably 3 to 7 ring carbon atoms, and may be unsubstituted or substituted, e.g. by Ci-Ct-alkyl or Ci-Ct-alkoxy. Cycloalkyl is, for example and preferably, cyclohexyl, cyclopentyl, methylcyclopentyl, or cyclopropyl, in particular cyclopropyl.
[0211] Acyl designates, for example, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, aryl- C1-C4- alkylcarbonyl, or heteroarylcarbonyl. Ci-C4-acyl is preferably lower alkylcarbonyl, in particular propionyl or acetyl. Ac stands for acetyl.
[0212] Hydroxyalkyl is especially hydroxy-(Ci-C4)-alkyl, preferably hydroxymethyl, 2-hydroxyethyl or 2-hydroxy-2 -propyl.
[0213] Haloalkyl is preferably fluoroalkyl, especially trifluoromethyl, 3,3,3-trifluoroethyl or pentafluoroethyl.
[0214] Halogen is fluorine, chlorine, bromine, or iodine.
[0215] Arylalkyl includes aryl and alkyl groups, e.g. benzyl, 1-phenethyl or 2-phenethyl. [0216] Heteroarylalkyl includes heteroaryl and alkyl as defined hereinbefore, and is e.g. 2-, 3- or 4- pyridylmethyl, 1- or 2-pyrrolylmethyl, 1-pyrazolylmethyl, 1-imidazolylmethyl, 2-(l-imidazolyl)ethyl or 3-(l-imidazolyl)propyl.
[0217] In substituted amino, the substituents are preferably those mentioned as substituents hereinbefore. In particular, substituted amino is alkylamino, dialkylamino, optionally substituted arylamino, optionally substituted arylalkylamino, lower alkylcarbonylamino, benzoylamino, pyridylcarbonylamino, lower alkoxycarbonylamino or optionally substituted aminocarbonylamino. [0218] Particular salts considered are those replacing the hydrogen atoms of a carboxylic acid function. Suitable cations are, e.g., sodium, potassium, calcium, magnesium or ammonium cations. [0219] In view of the close relationship between the novel compounds in free form and those in the form of their salts, including those salts that can be used as intermediates, for example in the purification or identification of the novel compounds, any reference to the free compounds herein is to be understood as referring also to the corresponding salts, and vice versa, as appropriate and expedient.
6. EXAMPLES
[0220] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.
6.1. Example 1. Synthesis of Compounds.
6.1.1. General Methods.
[0221] NMR spectra were recorded on a Bruker Avance DMX-500 (500.1 MHz) spectrometer. Assignment of 'H and 13C NMR spectra was achieved using 2D methods (COSY, HSQC, HMBC). Chemical shifts are expressed in ppm using residual CHCh, CHD2OD or HDO as references. Electron spray ionization mass spectra (ESI-MS) were obtained on a Waters micromass ZQ. The LC/HRMS analyses were carried out using an Agilent 1100 LC equipped with a photodiode array detector and a Micromass QTOF I equipped with a 4 GHz digital-time converter. Reactions were monitored by TLC using glass plates coated with silica gel 60 F254 (Merck) and visualized by using UV light and/or by charring with a molybdate solution (a 0.02 M solution of ammonium cerium sulfate dihydrate and ammonium molybdate tetrahydrate in aqueous 10% H2SO4). MPLC separations were carried out on a CombiFlash Companion or Rf from Teledyne Isco equipped with RediSep normal -phase or RP-18 reversed-phase flash columns. LC-MS separations were done on a Waters system equipped with sample manager 2767, pump 2525, PDA 2525 and micromass ZQ. Size -exclusion chromatography was performed on Bio-Gel® P-2 Gel (45-90 mm) from Bio-Rad (Reinach, Switzerland). All compounds used for biological assays are at least of 98% purity based on HPLC analytical results. Commercially available reagents were purchased from Sigma-Aldrich (Buchs, Switzerland), Acres (Geel, Belgium), Abcr (Germany), suppliers other than normal ones such as Glycosyn (New Zealand) and TCG (India) and et al. Solvents were purchased from Sigma-Aldrich (Buchs, Switzerland) or Acres Organics (Geel, Belgium) and were dried prior to use where indicated. Dichloromethane (DCM) was dried by fdtration over AI2O3 (Fluka, type 5016 A basic). Molecular sieves 4Ά were activated in vacuum at 500 °C for 1 h immediately before use.
[0222] Building blocks 10 [Z. Wang et al., J. Org. Chem. 2007, 72: 4209-6420], 11 [B. Sun et al.,
Sci. China. Chem. 2012, 55: 31-35], 14 [A. Berces et ak, Can. J. Chem. 2004, 82: 1157-1171], 15 [S. Ramadan et al., Org. Lett. 2017, 19: 4838-4841], 23a [B. Sun et al., Sci. China. Chem. 2012, 55: 31- 35], 23b [WO2018/ 167230], 24 [X.T. Zhang et al., Carbohydrate Research 2014, 388: 1-7], 28 [B. Ernst et al., WO2017/046172], 32 [B. Ernst et al., WO2017/046172] and 33 [B. Ernst et al., WO2017/046172] were prepared according to similar procedures in listed literatures.
6.1.2. Description of Synthetic Schemes.
[0223] Starting from known glycosyl donors (10 and 14) and glycosyl acceptors (11 and 15), two fully protected Gal-GalN disaccharides (12 and 16) were synthesized. By using the AgOTf/pTolSCl promoter system, donor 10 was pre-activated which resulted in high reactivity and selectivity therefore high yield for 12. The conventional TfOH/NIS condition was used for the preparation of 16. After removal of benzylidene protection, acetylation of diol intermediates yielded the Gal-GalN disaccharide building blocks (13 and 17) (Scheme 1).
[0224] Scheme 2 outlines the representative synthesis of the propionic acid analogues and the triflate building blocks (20 and 22), which involves the conversion of amino- to hydroxyl-group under Sandmeyer Reaction condition (NaNCE/ELSCE), benzylation of the carboxylic acid, and triflation. [0225] Compound 2 shares most of the structural features as natural GM1 epitope (1), except a tyramine moiety instead of amino-propyl glucoside (Part-I). Thus 1 and 2 were synthesized according to similar synthetic routes (Scheme 3). Sialic acid was firstly attached to the 3-position of the galactosides (23a-b) via NIS/TfOH sialylation conditions. Peracetylated sialyl donor (24) is known to have lower reactivity than common pyranosyl donors, due to the electron-withdrawing carbonyl group presenting at the anomeric carbon, therefore primarily reacts with the more reactive 3 -OH on the galactosyl acceptor (23a-b). Under AgOTf/pTolSCl-promoting pre -activation conditions, glycosylation between Gal-GalN donor (13) and sialosyl acceptors (25a-b) proceeded in 4-5 hours and gave fully-protected nat-GMl and GM1 mimetic (26a-b) in good yields. Acetyl- and benzoyl- protection of hydroxyls and NTroc were removed at once under basic condition at elevated temperature, and the acetamide was introduced to the 2-NH2 of GalN fragment with Ac20/MeOH/triethylamine. Both hydrogenation conditions, such as in-situ hydrogen generation with ammonium formate/palladium black (®natGMl 1), and palladium hydroxide/charcoal catalyzed hydrogenation (®GM1 mimetic 2), successfully removed the benzyl and Cbz protecting groups to yield the target oligosaccharides (1-2).
[0226] The synthesis of Neu5Ac (Part II)-replaced GM1 mimetics (3-9) was depicted in Scheme 4-6. Basically, the sialic acid moiety was replaced by a series of lactic acid analogs, which were introduced to the galactose acceptor (23a-b) via tin-mediated alkylation reaction by using -OTf as leaving group. Then Gal-GalN fragment (13 or 17) was coupled to the 4-position of galactoside or lactoside acceptors (29a-c, 34a-b, 37a-b) to give the fully-protected oligosaccharides (30a-c, 35a-b and 38a-b). After saponification, N-acetylation, hydrogenolysis, GM1 mimetics with replacement at Part-II (3-9) were obtained in good to excellent yields.
[0227] To synthesize the GM1 glyco-polymers, the monomeric GM1 analogues (1-9) were firstly reacted with thiobutyrolactone and triethylamine to attach a mercapto-butanamide linker ( >40-48) . The commercial linear or branched polylysines (linear 49-53 and branched 83-85, Scheme 7 and 8) were chloroacetylated, giving the activated polylysines - structurally represented as 54-58 for the linear and 86-88 for the branched polymers. Sub-stoichiometric amounts of the GM1 analogues were coupled to the lysine units through nucleophilic substitution of the chloride by sulfhydryl. In order to improve the aqueous solubility of the glycosylated-polylysine polymers, the remaining chloroacetamides were capped with thioglycerol. Nanofiltration with appropriate MW cut-off removed small-size impurities and the target glyco-polymers (59-82 and 89-97) were obtained as white solids after lyophilization.
Figure imgf000046_0001
[0228] Scheme 1. Synthesis of Gal-GalN disaccharide donors (14 and 18) via route (1) and (2). Reagents and conditions: a) AgOTf, pTolSCl, TTBP, 4A MS, CH3CN/DCM (1:10), -70 to -10°C, yield: 72%; b) i. TsOH, CH3CN/MeOH (1: 1), RT; ii. Ac20, pyridine, 0°C to RT, yield: 96%; c) TMSOTf, 4A MS, DCM/petrol ether (2: 1), -10 °C, yield: 40%; d) i. TsOH, water, MeOH, RT; ii. AC20. 4-DMAP, pyridine, 0°C to RT, yield: 81%.
Figure imgf000047_0001
L-3-Phenyllactic acid 21 22
[0229] Scheme 2. Synthesis of lactic acid analogs and triflate building blocks (20 and 22). Reagents and conditions: a) i. NaNC>2, H2SO4, water, 0°C to RT, yield: 55%; b) i. CS2CO3, MeOH/FbO (5: 1), RT; ii. BnBr, DMF, RT, yield over two steps: 44% for 19, 78% for 21; c) Tf20, 2,6-lutidine, DCM, - 15°C, yield: 78% for 20, 90% for 22.
Figure imgf000047_0002
[0230] Scheme 3. Synthesis of natural GMla epitope (1) and GM1 mimetic (2). Reagents and conditions: a) NIS, TfOH, 4A MS, CH3CN, -40°C to RT, yield: 47% for 25a, 58% for 25b; b) 13, AgOTf, pTolSCl, 4A MS, CH3CN/DCM (1: 10), -70 to 0°C, yield: 41% for 26a, 71% for 26b; c) i. IN NaOH(aq ), THF, 50°C; ii. Ac20, TEA, MeOH, RT, yield: quant for 27a, 79% for 27b; d) HCOONH4, Pd black, MeOH, water, RT, yield: 85%; e) H2 (gas), Pd(OH)2/C. 1,4-dioxane, water, RT, yield: 83%.
Figure imgf000048_0001
[0231] Scheme 4. Synthesis ofGMl mimetic (3-5). Reagents and conditions: a) i. Bu2SO. toluene, reflux; ii. triflate (20, 28, 22), CsF, DME, RT, yield: 65% for 29a, 72% for 29b, 54% for 29c; b) donor 13, AgOTf, pTolSCl, 4A MS, CH3CN/DCM (1: 10), -70 to 0°C, yield: 83% for 30a, 95% for 30b, 65% for 30c; c) i. IN NaOH(aq.), THF, 50°C; ii. Ac20. TEA, MeOH, RT, yield over two steps: 94% for 31a, 96% for 31b, 79% for 31c; d) e) ¾ (gas), Pd(OH)2/C, 1,4-dioxane or tBuOH, water, RT, yield: 81% for 3, 95% for 4, 80% for 5.
Figure imgf000049_0001
[0232] Scheme 5. Synthesis ofGMl mimetic (6-7). Reagents and conditions: a) i. BU2SO, toluene, reflux; ii. triflate (32, 33), CsF, DME, RT, yield: 55% for 34a, 84% for 34b; b) donor 17, TfOH, NIS, DCM, -20 to -5°C, yield: 26% for 35a, 26% for 35b; c) NaOMe, MeOH, RT, yield: quant for both 36a-b; d) H2 (gas), Pd(OH)2/C, tBuOH, DCM, AcOH, water, RT, yield: 24% for 6, 19% for 7.
Figure imgf000049_0002
[0233] Scheme 6. Synthesis ofGMl mimetic (8-9). Reagents and conditions: a) i. Bu2SO, toluene, reflux; ii. triflate (28, 33), CsF, DME, RT, yield: 55% for 37a, 93% for 37b; b) donor 17, TfOH, NIS, DCM, -20 to -5°C, yield: 45% for 38a, 18% for 38b; c) NaOMe, MeOH, RT, yield: quant for both 39a-b; d) H2 (gas), Pd(OH)2/C, tBuOH, AcOH, water, RT, yield: 40% for 8, 57% for 9.
Figure imgf000050_0001
[0234] Scheme 7. Synthesis of GM1 mimetic - linear polymer conjugates 59-82 with average degree of polymerization n = 100, 200, 400, 600 or 800. Reagents and conditions: a) GM1 mimetic (1-9), y- thiobutyrolactone, TEA, MeOH, RT, yield: quant.; b) Poly-L-lysines 49-53 with n = 100, 200, 400, 600 and 800 respectively, 2,6-lutidine, chloroacetic anhydride, DMF, 4°C, yield: 77-98%; c) i. DIPEA, DBU, water, DMF, RT; ii. 1-thioglycerol, TEA, water, DMF, RT, yield: 60-99% (12-37% GM1 -mimetic loading).
Figure imgf000051_0001
[0235] Scheme 8. Synthesis of GM1 mimetic - branched polymer conjugates 89-97 with average degree of polymerization (total of three branches) n = 150, 300 or 600. Reagents and conditions: a) GM1 mimetic 2, g-thiobutyrolactone, TEA, MeOH, RT, yield: quant.; b) Poly-L-lysines 83-85 with n = 150, 300 and 600 respectively, 2,6-lutidine, chloroacetic anhydride, DMF, 4°C, yield: 80-88%; c) i. DIPEA, DBU, water, DMF, RT; ii. 1-thioglycerol, TEA, water, DMF, RT, yield: 57-99% (13-37% GM1 -mimetic loading). [0236] Chemical Synthesis and structural characterization.
[0237] Compound 12: Glycosyl donor 10 (847 mg, 1.28 mmol) was dissolved in anhydrous DCM (15 mL) and then mixed with freshly activated 4Ά molecular sieves at room temperature for 30 min. The above mixture was then cooled down to -70 °C. AgOTf (988 mg, 3.8 mmol) in anhydrous CH3CN ( 1 mL) was added to the reaction mixture drop wise, after the mixture was stirred for 5 min, p-TolSCl (203 mg, 1.28 mmol) was added. The orange color of pTolSCl dissapeared immediately upon dropping into the vigorously stirred reaction mixture. A mixture of glycosyl acceptor 11 (702 mg, 1.28 mmol) and TTBP (318 mg, 1.28 mmol) in DCM/CH3CN (4: 1, 5 mL) was then added to the reaction mixture along the side of the reaction flask to ensure no temperature fluctuation. The reaction mixture was then gradually warmed up to -10 °C within 4 hours. The reaction was diluted with DCM (50 mL), and then filtered through Celite. The filtrate was washed twice with sat. NaHCCL (50 mL). The organic phase was collected and concentrated. The residue was purified on silica gel MPLC with 30-40% EtOAc/petrol ether to yield the product 12 as colorless oil (1 g, yield: 72%). TLC condition: Petrol ether / EtOAc (2: 1), Rf = 0.4. ¾ NMR (500 MHz, CDCL): d = 7.98 (d, J= 13 Hz, 2H, Ar-H), 7.57 (t, J= 1A Hz, 1H, Ar-H), 7.47 - 7.38 (m, 4H, Ar-H), 7.37 - 7.25 (m, 14H, Ar-H), 7.14 (dd, J= 29.5, 4.4 Hz, 6H, Ar-H), 6.91 (d, J= 7.9 Hz, 2H, Ar-H), 5.60 (dd, J= 9.8, 8.2 Hz, 1H), 5.33 (s, 1H), 5.20 (d, = 7.1 Hz, 1H), 5.15 (d, J= 10.0 Hz, 1H), 4.96 (d, .7= 11.7 Hz, 1H), 4.73 (d, J= 8.0 Hz, 1H), 4.66 (d, J= 12.1 Hz, 1H), 4.59 (dd, J= 12.0, 4.9 Hz, 2H), 4.50 - 4.39 (m, 3H), 4.36 (dd, J= 10.6, 2.8 Hz, 1H), 4.30 - 4.22 (m, 2H), 4.18 (d, J= 12.1 Hz, 1H), 3.94 (d, J= 2.5 Hz, 1H), 3.85 (d, J= 11.7 Hz, 1H), 3.68 - 3.49 (m, 4H), 3.39 (d, J= 24.8 Hz, 2H), 2.26 (s, 3H) ppm; 13C NMR (125 MHz, CDCL):
5 = 165.14, 153.73, 138.35, 137.96, 137.71, 137.42, 133.66, 133.11, 130.07, 129.88, 129.63, 128.69, 128.59, 128.50, 128.43, 128.38, 128.35, 128.03, 127.99, 127.93, 127.81, 127.77, 127.71, 126.47, 101.00, 100.64, 95.78, 84.30, 80.10, 75.57, 74.78, 74.53, 73.91, 73.83, 73.65, 72.41, 71.88, 71.55, 69.99, 69.26, 68.70, 51.41, 21.21 ppm; ESI-MS: m/z calcd for C57H56Ci3NNaOi2S [M + Na]+:
1106.25, found: 1106.29.
[0238] Compound 13: To a solution of compound 12 (790 mg, 0.73 mmol) in ClLCN/McOH (1: 1,
20 mL), TsOH monohydrate (277 mg, 1.46 mmol) was added at RT. The reaction mixture was stirred at RT and monitored by TLC. After 16 hours, the reaction was complete, and then neutralized with triethylamine (0.25 mL, 1.8 mmol) and concentrated under vacuum. The crude residue was dissolved in pyridine (4 mL), followed by addition of acetic anhydride at 0°C drop wise. The reaction mixture was then warmed up and stirred at room temperature overnight. The reaction was concentrated and the residue was purified on silica gel MPLC with 30-40% EtOAc/petrol ether to give the product 13 as white solid (750 mg, yield: 96%). TLC condition: Petrol ether / EtOAc (2: 1), Rf = 0.3. 'H NMR (500 MHz, CDCL): d = 8.05 - 7.99 (m, 2H), 7.64 - 7.56 (m, 1H), 7.47 (t, J= 7.8 Hz, 2H), 7.37 - 7.26 (m, 12H), 7.18 (dq, J = 6.9, 2.5 Hz, 1H), 7.14 - 7.08 (m, 4H), 7.03 (d, J= 8.0 Hz, 2H), 5.53 (dd, J = 10.0, 7.9 Hz, 1H), 5.36 (d, J= 3.3 Hz, 1H), 5.09 (d, J= 13 Hz, 1H), 5.05 (d, J= 10.3 Hz, 1H), 4.94 (d, J = 11.6 Hz, 1H), 4.62 - 4.55 (m, 3H), 4.54 - 4.37 (m, 4H), 4.31 (dd, J= 10.3, 3.2 Hz, 1H), 4.12 (dd, J= 11.7, 4.4 Hz, 1H), 4.07 - 3.95 (m, 2H), 3.90 (d, J= 12.1 Hz, 1H), 3.75 (dd, J= 7.7, 4.5 Hz, 1H), 3.69 - 3.49 (m, 4H), 3.33 (d, J= 7.4 Hz, 1H), 2.30 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H) ppm; 13C NMR (125 MHz, CDC ): d = 170.49, 170.01, 165.02, 153.45, 138.52, 138.06, 137.86, 137.42,
133.08, 132.80, 130.12, 129.87, 129.55, 128.87, 128.49, 128.31, 128.22, 127.95, 127.93, 127.87, 127.73, 127.70, 127.52, 101.35, 95.58, 85.48, 79.50, 75.26, 74.42, 73.63, 73.61, 72.18, 71.79, 71.54, 69.10, 68.09, 63.05, 52.99, 21.11, 20.75, 20.69 ppm; ESI-MS: m/z: calcd for CsrHseCfeNNaOuS [M + Na]+: 1102.24, found: 1102.22.
[0239] Compound 16: Compound 14 (500 mg, 0.79 mmol), 15 (285 mg, 0.55 mmol) and freshly activated 4Ά molecular sieves were mixed in DCM/petrol ether (10 mL, 2: 1). The mixture was cooled to -10°C, and then added TMSOTf (17 mg, 0.08 mmol) drop wise. The reaction mixture was stirred at -10°C for 10 min then quenched with triethylamine. The reaction mixture was fdtered and concentrated, and the residue was purified on silica gel MPLC with 30-40% EtOAc/petrol ether to give the product 16 as white solid (710 mg, yield: 40%). TLC condition: Petrol ether / EtOAc (3:2),
Rf = 0.59. ¾ NMR (500 MHz, CDCE): d = 7.57 - 7.49 (m, 2H), 7.48 - 7.40 (m, 2H), 7.39 - 7.18 (m, 17H), 7.01 (d, J = 7.6 Hz, 2H), 6.84 (d, J= 7.1 Hz, 2H), 5.48 (s, 1H), 5.36 - 5.23 (m, 2H), 4.90 (d, J = 10 Hz, 1H), 4.63 (d, J= 12.2 Hz, 1H), 4.60 - 4.48 (m, 3H), 4.48 - 4.37 (m, 3H), 4.37 - 4.27 (m, 2H), 3.97 - 3.85 (m, 2H), 3.77 - 3.66 (m, 1H), 3.62 - 3.50 (m, 3H), 3.43 (q, J= 1.5 Hz, 1H), 3.36 (dd, J = 10.0, 2.8 Hz, 1H), 2.31 (s, 3H), 1.90 (s, 3H) ppm; ESI-MS: m/z calcd for CstTfeCLNNaOnS [M + Na]+: 1002.22 found: 1002.25.
[0240] Compound 17: Compound 16 (710 mg, 0.72 mmol) was dissolved in MeOH (20mL) and p- toluenesulfonic acid monohydrate (204 mg, 1.07 mmol) with water (250pmL) was added at RT. The solution was stirred overnight and then quenched with triethylamine (0.5 mL). The reaction mixture was vacuum dried and the residue was treated with pyridine (10 mL). To the above mixture, AC2O (1.09 g, 10.7 mmol) and 4-DMAP (8.7 mg, 0.07 mmol) was added at 0°C. The reaction mixture was stirred at RT overnight, concentrated and the residue was purified on silica gel MPLC with acetone/toluene to give the product 17 as white solid (570 mg, yield: 81%). TLC condition: Petrol ether / EtOAc (3:2), Rf= 0.4. ¾ NMR (500 MHz, CDCI3): d = 7.46 - 7.38 (m, 2H), 7.35-7.24 (m, 14H), 7.10 (d, J= 7.9 Hz, 2H), 6.93 (d, J= 7.6 Hz, 1H), 5.39 (d, J= 3.3 Hz, 1H), 5.23 (dd, J= 10.1, 7.9 Hz, 1H), 5.07 (d, J= 10.3 Hz, 1H), 4.89 (d, J= 11.6 Hz, 1H), 4.62 (d, J= 12.3 Hz, 1H), 4.56 - 4.40 (m, 5H), 4.37 (dd, J= 10.4, 3.3 Hz, 1H), 4.11 (dd, J= 11.7, 4.9 Hz, 1H), 4.05 (dd, J= 11.7, 7.6 Hz, 1H), 3.89 (d, J= 2.7 Hz, 1H), 3.81 - 3.71 (m, 2H), 3.60 - 3.50 (m, 2H), 3.50 (t, J= 6.4 Hz, 1H), 3.38 (dd, J= 10.1, 2.8 Hz, 1H), 2.34 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H) ppm; 13C NMR (125 MHz, CDCh): d = 170.51, 169.90, 169.70, 161.52, 138.53, 138.41, 137.78, 137.64, 133.24, 129.72, 128.49, 128.47, 128.44, 128.21, 127.98, 127.90, 127.83, 127.54, 127.51, 100.52, 85.25, 79.96, 75.35, 74.33, 73.96, 73.85, 73.56, 72.20, 71.77, 70.91, 69.07, 68.32, 62.74, 53.58, 21.21, 21.18, 20.77, 20.67 ppm; ESI-MS: m/ calcd for C47H52Cl3NNaOi3S [M + Na]+: 998.21, found: 998.41.
[0241] Compound 18: To a stirring mixture of L-3-cyclohexylalanine (1 g, 5.8 mmol) and 1M H2SO4 (10 mL) at 0 °C, NaNCL aqueous solution (6 mL, 0,4 g/mL) was added drop wise over 30 min. After stirring at 0 °C for 1 hour, the reaction mixture was warmed up to RT and stirred overnight. The reaction mixture was diluted with DCM (80 mL) and extracted with brine (50 mL c 3). The organic layer was collected, dried over Na2S04, filtered and concentrated to give the product as colorless oil, which was used directly in the next step. ESI-MS: m/ calcd for CqHigNaCL [M + Na]+: 195.10, found: 195.17.
[0242] General procedure for synthesis of compound 19 and 21: (S)-3-cyclohexyl-2- hydroxypropanoic acid (18) or L-3-phenyllactic acid (300-500 mg, 1 equiv.) was dissolved in MeOH (5 mL) and then mixed with 1.5N CS2CO3 aqueous solution (1 equiv.). After stirring at RT for 1 hour, the reaction mixture was dried under vacuum. The residue was mixed with DML (5 mL) and BnBr (1.1 equiv.) was added drop wise. After stirring at RT overnight, the reaction mixture was extracted with EtOAc (30 mL) and water (30 mL). The organic phase was collected, dried over Na2S04, and concentrated. The residue was purified on silica gel MPLC with 20% EtOAc/petrol ether to give the product as colorless oil.
[0243] Compound 19: 240 mg (44%). ‘HNMR (500 MHz, CDCI3): d = 7.44 - 7.31 (m, 5H, Ph), 5.20 (s, 2H, CLCPh), 4.37 - 4.23 (m, 1H, OH), 2.67 (d, J= 5.5 Hz, 1H, -C770H), 1.88 - 1.47 (m, 8H), 1.34 - 1.08 (m, 3H), 1.02 - 0.82 (m, 2H) ppm; ESI-MS: m/r calcd for CigH22Na03 [M + Na]+: 285.15, found: 285.49.
[0244] Compound 21: 363 mg (78%). ‘HNMR (500 MHz, CDCI3): d = 7.44 - 7.27 (m, 5H), 7.26 - 7.16 (m, 3H), 7.13 (dd, J= 7.5, 1.5 Hz, 2H), 5.15 (d, J= 3.3 Hz, 2H), 4.46 (q, J= 5.0 Hz, 1H), 3.10 (dd, J= 13.9, 4.7 Hz, 1H), 2.96 (dd, J= 13.9, 6.6 Hz, 1H), 2.84 (d, J= 5.1 Hz, 1H) ppm; ESI-MS: m/r calcd for Ci6Hi6Na03 [M + Na]+: 279.10, found: 279.17.
[0245] General procedure for synthesis of compound 20 and 22: A solution of 19 or 21 (300-400 mg, 1 equiv.) and 2,6-lutidine (2 equiv.) in DCM (6 mL) was cooled to -15°C and triflic acid anhydride (1.5 equiv. in 4 mL of DCM) was added drop wise. After stirring for 40 min, cooling bath was removed; reaction mixture was stirred at RT. After 0.5 hour, the reaction mixture was quenched with ice-cold sat. NaaCCL ( q.) (20 mL) and brine (20 mL). The organic layer was dried over Na2S04, filtered and concentrated. The residue was filtered through silica gel cartridge with an elution of 5% EtOAc/petrol ether to give the product as colorless oil (20 and 22). Due to the instability of triflate products, the product was collected, vacuum dried and then used immediately in next step.
[0246] General procedure for the synthesis of 25a-b: A mixture of glycosyl acceptor 23a or 23b (1 - 3 g, 1 equiv.), glycosyl donor 24 (1 - 3 g, 1.2 equiv.), NIS (0.6 - 2.7 g, 2.4 equiv.) and freshly activated 4Ά molecular sieves in anhydrous CH3CN (20 - 40 mL) was stirred at RT for 30 min. The mixture was then cooled to -30°C, and TfOH (25-80 pL. 0.2 equiv.) was added drop wise. The reaction was stirred at -30 °C for 4 hours, and then slowly warmed up to RT overnight. The reaction was quenched with triethylamine (100 pL). and then fdtered and concentrated. The residue was dissolved in EtOAc (50 - 100 mL) and washed with sat. Na2S2C>3(aq) (50 - 100 mL). The organic phase was collected, concentrated, and the residue was purified on silica gel MPLC with 5 - 10% iPrOH/(petrol ether/DCM 2: 1) to give the product (25a-b) as white or light yellow solid.
[0247] Compound 25a: 1.92 g (47%) as light yellow solid. ‘HNMR (500 MHz, CDCh): d = 7.37 - 7.33 (m, 2H), 7.30 - 7.13 (m, 23 H), 6.50 (d, J= 10 Hz, 1 H, NH), 5.38 - 5.33 (m, 1 H), 5.26 - 5.21 (m, 1 H), 5.05 - -4.97 (m, 1 H), 4.94 (d, J= 10.0 Hz, 1 H), 4.77 (d, J= 10.0 Hz, 1 H), 4.74 - 4.61 (m, 4 H), 4.53 (d, J= 10.0 Hz, 1 H), 4.50 - 4.36 (m, 3 H), 4.33 - 4.27 (m, 2 H), 4.27 - 4.21 (dd, J= 15.0, 5.0 Hz, 1H), 4.18 - 4.13 (dd, J= 15.0, 5.0Hz, 1 H), 4.01 - 4.37 (dd, J= 10.0, 5.0 Hz, 1 H), 3.96 - 3.85 (m, 3 H), 3.79 (d, J= 5.0 Hz, 1 H), 3.73 (s, 3 H), 3.70 - 3.67 (m, 2 H), 3.66 - 3.60 (dd, J= 11.5, 9.0 Hz, 1 H), 3.60 - 3.42 (m, 6 H), 3.36-3.28 (m, 4 H), 2.54 - 2.49 (dd, J= 10.0 Hz, 5.0 Hz, 1 H), 2.11 (s, 3 H, 2.05 (s, 3 H), 1.97 (t, J= 10.0 Hz, 1 H), 1.96 (s, 3 H), 1.91 (s, 3 H), 1.85 (s, 3 H), 1.84 - 1.77 (m, 2 H) ppm; 13C NMR (125 MHz, CDCh): d = 172.9, 171.1, 171.0, 170.0, 166.7, 153.9, 139.2, 138.7, 138.4, 138.3, 138.2, 128.6, 128.58, 128.3, 128.2, 128.1, 128.0, 127.93, 127.91, 127.88, 127.8, 127.7, 127.6, 103.8, 102.6, 99.3, 83.0, 81.9, 78.1, 77.7, 76.2, 75.6, 75.5, 75.3, 75.2, 74.5, 73.7, 73.5, 72.6, 69.3, 66.7, 63.5, 59.6, 53.1, 48.5, 29.5, 25.0, 24.4, 21.4, 21.0 ppm; ESI-MS: m/z: calcd for C7oH84N4Na023 [M + Na]+: 1371.54, found: 1371.58.
[0248] Compound 25b: 0.96 g (58%) as white solid. ¾ NMR (500 MHz, CDCh): d = 7.45 - 7.22 (m, 18H), 7.14 (s, 1H), 7.04 (d, J= 7.6 Hz, 1H), 7.00 - 6.89 (m, 4H), 5.42 (ddd, J= 8.4, 5.9, 2.7 Hz, 1H), 5.31 (dd, J= 8.1, 2.1 Hz, lH), 5.18 (s, 2H), 5.16 (d, J= 9.7 Hz, 1H), 5.01 (s, 1H), 4.93 - 4.79 (m, 3H), 4.58 - 4.55 (m, 2H), 4.46 - 4.34 (m, 2H), 4.36 - 4.25 (m, 2H), 4.09 - 3.92 (m, 4H), 3.86 - 3.74 (m, 8H), 3.49 - 3.32 (m, 2H), 2.82 - 2.66 (m, 2H), 2.57 (dd, J= 13.0, 4.6 Hz, 1H), 2.10 (s, 2H), 2.04 (brs, 4H), 2.00 (s, 3H), 1.99 (s, 3H), 1.95 (s, 2H) ppm; ESI-MS: m/z calcd for [M + Na]+: 1199.46, found: 1199.53.
[0249] General procedure of the preactivation-based glycosylation reactions for the synthesis of 26a- b, 30a-c: Glycosyl donor 13 (70 - 200 mg, 1 equiv.) and glycosyl acceptor 25a/b, 29a/b/c (1.5 equiv.) were dissolved in anhydrous DCM (20 mL) and mixed with freshly activated 4Ά molecular sieves at room temperature for 30 min. The above mixture was then cooled down to -70 °C. AgOTf (3 equiv.) in anhydrous CH3CN (2 mL) was added to the reaction mixture. After stirring for 5 min, p-TolSCl (1 equiv.) was added drop wise. The reaction mixture was then gradually warmed up to 0 °C within 5 hours. The reaction mixture wasdiluted with DCM (80 mL) and filtered through Celite. The filtrate was concentrated and the residue was purified on silica gel MPLC with 0-6% iPrOH /(PE/DCM (2: 1)) to yield the product (29a-b, 33a-c, 43). [0250] Compound 26a: 176 mg (41%) as colorless oil. ¾ NMR (500 MHz, CDCI3): d = 7.95 (s, 2 H), 7.54 - 6.99 (m, 45 H), 5.52 - 5.46 (t, 1 H), 5.44 (d, J= 7.0 Hz, 1H), 5.39 (d, J= 3.0 Hz, 1H), 5.25
- 5.20 (m, 2 H), 5.16 (d, J= 10 Hz, 2 H), 5.05 - 5.00 (m, 1 H), 4.94 (d, J= 10 Hz, 2 H), 4.80 - 4.76 (m, 3 H), 4.75 - 4.65 (m, 3 H), 4.63 - 4.52 (m, 4 H), 4.51 - 4.37 (m, 5 H), 4.36 - 4.30 (m, 2 H), 4.26
- 4.08 (m, 2 H), 4.05 - 3.76 (m, 11 H), 3.72 - 3.55 (m, 8 H), 3.54 - 3.47 (t, 2H), 3.45 - 3.39 (m, 2H), 3.38 - 3.36 (m, 5 H), 2.11 - 1.97 (m, 8 H), 1.96 (s, 6H), 1.89 (s, 3 H), 1.88 (s, 3 H), 1.87 - 1.70 (m, 5 H); 13C NMR (125 MHz, CDCI3): d = 170.9, 170.5, 170.47, 170.2, 168.7, 165.3, 162.2, 139.11, 139.1, 139.0, 138.9, 138.8, 138.5, 138.0, 133.2, 130.5, 130.1, 129.3, 128.6, 128.57, 128.5, 128.49, 128.4, 128.36, 128.2, 128.15, 127.8, 127.77, 127.7, 127.6, 127.5, 126.6, 126.5, 103.7, 102.7, 100.9, 100.6, 100.5, 83.1, 82.5, 81.9, 80.3, 76.8, 76.0, 75.6, 75.5, 75.45, 75.4, 74.0, 73.4, 73.3, 73.1, 73.0, 72.9,
72.3, 71.8, 70.2, 69.4, 69.1, 68.5, 67.7, 67.6, 67.2, 66.7, 66.4, 62.4, 53.1, 51.8, 48.6, 29.9, 25.5, 21.7, 21.0, 20.9, 20.8 ppm; ESI-MS: m/z calcd for CnvH CEN NaCEv [M + Na]+: 2326.76, found: 2327.00.
[0251] Compound 26b: 225 mg (71%) as colorless oil. ¾ NMR (500 MHz, CDCI3): d = 8.06 (d, J=
7.7 Hz, 2H), 8.01 (s, 1H), 7.60 (t, J= 1A Hz, 1H), 7.50 - 7.43 (m, 3H), 7.39 - 7.27 (m, 22H), 7.25 -
7.20 (m, 4H), 7.17 - 7.09 (m, 6H), 7.05 - 6.87 (m, 4H), 5.50 (dd, J= 10.1, 7.8 Hz, 1H), 5.40 - 5.30 (m, 4H), 5.30 - 5.24 (m, 1H), 5.18 (s, 3H), 5.01 - 4.84 (m, 5H), 4.71 - 4.54 (m, 5H), 4.52 - 4.33 (m, 9H), 4.25 - 4.15 (m, 2H), 4.07 - 3.83 (m, 7H), 3.77 - 3.56 (m, 9H), 3.53 - 3.33 (m, 4H), 2.94 (s, 3H), 2.87 (s, 3H), 2.82 - 2.62 (m, 3H), 2.51 (dd, J= 13.2, 4.7 Hz, 1H), 2.03 (brs, 4H), 1.95 (d, J= 5.8 Hz, 6H), 1.91 (s, 6H) ppm; 13C NMR (125 MHz, CDCh): d = 170.78, 170.42, 170.31, 170.15, 169.76, 168.40, 164.97, 162.57, 153.72, 138.83, 138.56, 138.35, 137.86, 137.76, 137.51, 136.76, 132.97,
130.33, 129.93, 129.64, 128.56, 128.52, 128.49, 128.40, 128.34, 128.30, 128.23, 128.18, 128.10,
128.01, 127.89, 127.85, 127.72, 127.55, 127.50, 127.40, 127.36, 127.28, 117.06, 101.14, 99.70, 98.40,
95.99, 79.50, 74.98, 74.41, 73.71, 73.51, 73.46, 72.40, 72.26, 71.72, 70.53, 69.73, 69.00, 68.20, 64.39, 62.50, 62.07, 52.88, 50.97, 49.34, 36.48, 31.44, 29.69, 25.35, 23.87, 23.17, 21.15, 20.79, 20.63, 20.60 ppm; ESI-MS: m/z: calcd for CnoH CENsNaCEr [M + Na]+: 2154.67, found: 2154.38.
[0252] Compound 30a: 132 mg (83%) as colorless oil. ¾ NMR (500 MHz, CDCI3): d = 8.08 (d, J =
7.8 Hz, 2H), 7.60 - 7.52 (m, 1H), 7.46 (t, J= 7.7 Hz, 2H), 7.40 - 7.11 (m, 40H), 6.99 (d, J= 7.9 Hz, 1H), 6.87 (s, 3H), 5.84 (d, = 8.0 Hz, 1H), 5.55 (t, = 9.0 Hz, 1H), 5.36 (d, = 3.6 Hz, lH), 5.17 (s, 2H), 5.06 (d, J= 8.4 Hz, 1H), 5.00 - 4.90 (m, 3H), 4.84 - 4.56 (m, 7H), 4.53 - 4.21 (m, 10H), 4.18 - 4.03 (m, 1H), 4.03 - 3.89 (m, 2H), 3.83 (t, J= 8.5 Hz, 1H), 3.77 - 3.48 (m, 10H), 3.46 - 3.31 (m, 2H), 2.81 - 2.61 (m, 2H), 2.13 - 2.03 (m, 4H), 1.95 (s, 3H), 1.69 - 1.48 (m, 7H), 1.17 - 0.98 (m, 4H), 0.94
- 0.70 (m, 3H) ppm; 13C NMR (125 MHz, CDCh): d = 170.48, 170.20, 165.13, 155.87, 138.71,
138.51, 138.27, 137.98, 137.81, 137.65, 136.81, 135.40, 132.93, 130.55, 130.01, 129.72, 128.60,
128.56, 128.50, 128.43, 128.40, 128.36, 128.26, 128.25, 128.01, 127.96, 127.92, 127.89, 127.85,
127.73, 127.66, 127.58, 127.46, 117.13, 101.37, 100.08, 100.00, 96.21, 79.65, 79.25, 77.98, 74.56, 74.40, 74.12, 73.70, 73.61, 73.25, 72.59, 71.85, 71.78, 71.53, 69.58, 69.37, 68.42, 67.34, 67.20, 66.43, 62.75, 54.97, 50.99, 48.29, 40.42, 34.05, 33.52, 33.19, 26.22, 26.15, 20.74, 20.71 ppm; ESI-MS: m/r calcd for CioeH CyNfeNaOar [M + Na]+: 1925.66 , found: 1925.76.
[0253] Compound 30b: 180 mg (95%) as colorless oil. 'HNMR (400 MHz, CDCh): d = 8.06 (d, J= 7.7 Hz, 1H), 7.62 - 7.55 (m, 1H), 7.50 - 7.44 (m, 2H), 7.40 - 7.09 (m, 40H), 7.02 - 6.83 (m, 4H),
5.54 (t, J= 8.9 Hz, 1H), 5.34 (d, J= 3.5 Hz, 1H), 5.27 (d, J= 7.3 Hz, 1H), 5.16 (d, J= 15.7 Hz, 4H), 5.07 (d, J= 12.1 Hz, 2H), 4.97 (d, J= 11.8 Hz, 2H), 4.76 (s, 1H), 4.68 - 4.56 (m, 5H), 4.51 - 4.30 (m, 10H), 4.21 (d, J = 12.0 Hz, 2H), 3.97 (dd, J= 3.0, 1.0 Hz, 1H), 3.92 (dd, J = 11.4, 6.7 Hz, 1H), 3.82 - 3.29 (m, 11H), 2.82 - 2.62 (m, 2H), 2.07 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.58 (d, J= 13.8 Hz, 8H), 1.05 (d, J= 8.4 Hz, 3H), 0.85 - 0.67 (m, 2H) ppm; 13C NMR (101 MHz, CDCI3): d = 173.56, 170.64, 170.26, 165.11, 155.91, 138.78, 138.48, 138.04, 137.88, 137.68, 136.88, 135.42, 133.01, 130.04, 129.77, 128.82, 128.78, 128.67, 128.62, 128.59, 128.50, 128.42, 128.31, 128.10, 128.05, 128.02, 127.95, 127.79, 127.73, 127.67, 127.52, 117.13, 102.05, 81.78, 79.65, 75.14, 74.41, 74.13, 73.67, 72.51, 71.83, 71.75, 71.30, 69.48, 68.46, 67.39, 66.79, 62.58, 55.11, 51.05, 48.36, 41.29, 34.22, 33.65, 33.60, 33.09, 26.35, 26.03, 20.90, 20.83 ppm; ESI-MS: m/r calcd for CioeH CWNfeNaOar [M + Na]+: 1925.66, found: 1925.68.
[0254] Compound 30c: 80 mg (65%) as colorless oil. 'HNMR (500 MHz, CDCI3): d = 8.07 (d, J= 7.2 Hz, 2H), 7.56 (t, J= 7.4 Hz, 1H), 7.46 (t, J= 7.6 Hz, 2H), 7.39 - 7.03 (m, 45H), 6.97 (s, 1H), 6.85 (s, 3H), 5.82 (d, J= 7.8 Hz, 1H), 5.53 (t, J= 8.8 Hz, 1H), 5.29 (s, 1H), 5.17 (s, 2H), 5.02 - 4.89 (m, 3H), 4.78 - 4.54 (m, 9H), 4.53 - 4.30 (m, 7H), 4.07 (dd , J= 11.0, 5.2 Hz, 1H), 4.04 - 3.87 (m, 2H), 3.81 (s, 1H), 3.75 - 3.49 (m, 7H), 3.48 - 3.28 (m, 5H), 3.04 (dd , J= 14.1, 5.0 Hz, 1H), 2.95 (d, J= 7.4 Hz, 1H), 2.82 - 2.59 (m, 2H), 2.03 (d, J= 5.4 Hz, 4H), 2.00 (s, 3H) ppm; 13C NMR (125 MHz, CDCh): d = 170.53, 170.19, 155.80, 138.63, 137.92, 137.77, 136.78, 135.16, 132.89, 129.98, 129.66, 129.44, 128.58, 128.51, 128.35, 128.30, 128.24, 128.07, 127.94, 127.87, 127.71, 127.54, 127.49, 117.08, 102.01, 101.15, 99.97, 96.29, 79.68, 74.40, 73.74, 73.62, 71.84, 62.63, 50.97, 49.23, 48.27, 39.21, 39.07, 20.79, 20.69, 0.03 ppm; ESI-MS: m/ calcd for CioeHiovCySfeNaOMlM + Na]+:
1919.62, found: 1919.63.
[0255] General procedure for the synthesis of 27a-b, 31a-c: Compound 26a/b (200 mg, 1 equiv.) was dissolved in a mixture of IN NaOH (aq.)/THF (5: 1, 12 mL) and stirred at RT. The reaction progress was monitored by TLC (DCM/MeOH 12:1). After the starting material was fully consumed, THF was removed by evaporation, and the residue was extracted with DCM (50 mL) and water (30 mL). The organic layer was concentrated to dryness to give the deacetylation intermediate. The intermediate was then dissolved in a mixture of methanol (6 mL), triethylamine (20 equiv.) and acetic anhydride (20 equiv.). The reaction mixture was stirred at RT for 4 hours, and then concentrated. The residue was purified by passing through a silica cartridge with elution of 10-20% MeOH/DCM. The product (27a-b, 31a-c) was collected, concentrated and used directly in next hydrogenation step. [0256] Compound 1: Compound 27a (20 mg, 0.011 mmol) was dissolved in MeOH/water (1: 1, 0.8 mL) to form a white slurry mixture after sonication for a few seconds. Pd black (10 mg) and ammonium formate (10 mg) was added to above mixture and then heated at 50 °C. The reaction was monitored by LC-MS, and after 2.5 hours complete conversion from 27a to compound 1 was observed. The reaction mixture was filtered, then concentrated and lyophilized from water to give the product as white solid (10 mg, yield: 85%). ‘HNMR (500 MHz, D2O): d = 4.71 (d, 1H, J= 8.5 Hz), 4.50 - 4.43 (m, 3 H), 4.12 - 4.05 (m, 3 H), 4.01 - 3.89 (m, 3 H), 3.85 (d, 1H, J= 3.5 Hz), 3.81 (dd,
1H, J= 13.0, 4.0 Hz), 3.79 - 3.65 (m, 14 H), 3.65 - 3.50 (m, 7 H), 3.46 - 3.41 (m, 2 H), 3.32 -.23 (m, 2 H), 3.07 (t, 2 H, J = 6.6 Hz), 2.60 (dd, 1 H, J= 12.5, 4.0 Hz), 1.97 (s, 3 H), 1.94 (s, 3 H), 1.94 (t, 1
H , J= 12 Hz), 1.90 - 1.84 (m, 2 H).; 13C NMR (125 MHz, D20): d = 181.6, 175.1, 174.9, 174.2,
104.8, 102.7, 102.6, 102.2, 101.8, 80.5, 78.7, 77.2, 75.0, 74.9, 74.5, 74.2, 73.2, 72.8, 72.6, 72.4, 70.8,
68.8, 68.7, 68.1, 68.0, 63.0, 61.2, 61.1, 60.8, 60.2, 51.7, 51.3, 37.7, 27.1, 23.4, 22.7, 22.2 ppm;
HRMS: m z: calcd for C4oH69N3Na029 [M + Na]+: 1078.3914, found: 1078.3914.
[0257] General procedure of the Pd(OH)2/C catalyzed hydrogenation reactions for the synthesis of GM1 mimetics (2-9): Compound 27b, 31a/b/c was dissolved in a mixture of 1,4-dioxane/water (1: 1 for 27b, 2: 1 for 31a and 31c) or in a mixture of tBuOH/water (7:3 for 31b). Pd(OH)2/C (20-100% weight of the glycan) was added and the reaction mixture was stirred under ¾ atmosphere (balloon) at RT. The progress of reaction was monitored by TLC and LC-MS. After 16-24 hours, the reaction mixture was filtered through Celite and the filtrate was concentrated under vacuum. The residue was dissolved with DI water, filtered through 0.22 pm PTFE filter, and lyophilized from water to give the product (2-9).
[0258] Compound 2: 53 mg (83%) as white solid. ¾ NMR (500 MHz, D20): d = 7.23 (d, J= 8.6 Hz, 2H), 7.06 (d, J= 8.6 Hz, 2H), 5.07 (d, J= 7.9 Hz, 1H), 4.49 (d, J= 7.8 Hz, 1H), 4.19 (dd, J= 9.8, 2.9 Hz, 1H), 4.11 (dd, J= 10.9, 2.7 Hz, 2H), 4.00 (dd, J= 10.7, 8.7 Hz, 1H), 3.89 - 3.60 (m, 15H), 3.60 - 3.51 (m, 4H), 3.46 (ddd, J= 9.8, 4.7, 2.8 Hz, 2H), 3.19 (t, J= 7.1 Hz, 2H), 2.90 (t, J= 7.2 Hz, 2H), 2.68 - 2.58 (m, 1H), 1.98 (s, 3H), 1.97 (s, 3H), 1.88 (t, J= 11.9 Hz, 1H) ppm; 13C NMR (125 MHz, D2O): d = 175.04, 174.80, 174.08, 155.54, 130.20, 117.21, 104.72, 102.56, 101.58, 100.26, 80.21, 76.72, 74.88, 74.37, 74.11, 73.06, 72.49, 72.28, 70.67, 69.43, 68.65, 68.58, 68.04, 67.88, 62.84, 61.08, 60.93, 60.16, 51.58, 51.21, 40.63, 37.01, 31.97, 22.59, 22.04 ppm; HRMS: m/r. calcd for C39H6iN3Na024 [M + Na]+: 978.3537, found: 978.3540.
[0259] Compound 3: 42 mg (81%) as white solid. ¾ NMR (500 MHz, D20): d = 7.22 (t, J= 8.4 Hz, 2H), 7.06 (d, J= 8.7 Hz, 2H), 4.99 (d, J= 7.8 Hz, 1H), 4.75 (d, J= 8.5 Hz, 1H), 4.42 (d, J= 7.7 Hz, 1H), 4.28 (d, J= 2.4 Hz, 1H), 4.14 (d, J= 3.1 Hz, 1H), 4.12 - 4.06 (m, 1H), 3.99 (dd, J= 10.9, 8.5 Hz, 1H), 3.85 (d, J= 3.3 Hz, 1H), 3.83 - 3.64 (m, 9H), 3.64 - 3.55 (m, 3H), 3.52 (dd, J= 10.0, 2.5 Hz, 1H), 3.47 (dd, J= 9.9, 7.7 Hz, 1H), 3.38 - 3.29 (m, 2H), 2.97 (t, J= 7.7 Hz, 2H), 2.04 (s, 3H),
I.84 - 1.49 (m, 7H), 1.43 - 1.30 (m, 1H), 1.23 - 1.07 (m, 3H), 0.95 (dt, J= 11.7, 5.7 Hz, 2H) ppm; 13C NMR (125 MHz, D20): d = 181.43, 174.95, 155.69, 130.80, 130.39, 130.18, 130.14, 130.08, 117.13, 104.74, 101.45, 100.81, 81.35, 80.05, 79.83, 75.09, 74.89, 74.77, 72.79, 72.46, 70.64, 69.81,
68.60, 68.06, 61.00, 60.89, 60.48, 58.54, 51.60, 50.12, 42.76, 40.88, 40.61, 34.11, 33.35, 32.79, 32.67,
31.90, 29.37, 25.99, 22.46 ppm; HRMS: m/r calcd for C37H59N2O18 [M + H]+: 819.3757, found: 819.3757.
[0260] Compound 4: 20 mg (95%) as white solid. ¾ NMR (500 MHz, D20): d = 7.29 - 7.15 (m, 2H), 7.10 - 6.96 (m, 2H), 4.95 (d, = 7.8 Hz, 1H), 4.74 (d, = 8.6 Hz, 1H), 4.45 (d, = 7.8 Hz, 1H), 4.20 (d, J= 2.7 Hz, 1H), 4.08 (d, J= 3.2 Hz, 1H), 4.03 - 3.93 (m, 2H), 3.87 - 3.79 (m, 2H), 3.79 - 3.52 (m, 11H), 3.50 - 3.41 (m, 2H), 3.17 (t, J= 7.2 Hz, 2H), 2.88 (t, J= 7.2 Hz, 2H), 2.05 (s, 3H), 1.80 - 1.39 (m, 7H), 1.27 - 1.02 (m, 3H), 0.97 - 0.82 (m, 2H) ppm; 13C NMR (125 MHz, D20): d = 81.18, 174.91, 155.75, 131.16, 130.18, 117.20, 104.73, 102.08, 100.78, 80.70, 80.39, 80.29, 74.91, 74.56, 74.41, 74.39, 72.49, 70.67, 70.24, 68.59, 68.04, 60.95, 60.36, 51.40, 40.61, 40.33, 33.24, 33.11, 33.03, 31.92, 26.11, 25.88, 22.65 ppm; HRMS: m/r calcd for CsvHssNaNaOis [M + Na]+: 841.3577, found: 841.3572
[0261] Compound 5: 23 mg (80%) as white solid. 'HNMR (500 MHz, D20): d = 7.49 - 7.42 (m, 4H), 7.36 (ddd, J = 8.4, 5.7, 2.7 Hz, 1H), 7.21 (d, J= 8.7 Hz, 2H), 7.08 - 6.99 (m, 2H), 4.97 (d, J=
7.7 Hz, 1H), 4.54 (dd, J= 9.7, 3.1 Hz, 1H), 4.34 (d, = 7.8 Hz, 1H), 3.98 (d, = 3.1 Hz, 1H), 3.89 (dd, = 6.1, 2.7 Hz, 2H), 3.83 - 3.57 (m, 11H), 3.53 (dd, = 10.0, 2.5 Hz, 1H), 4.34 (d, = 7.8 Hz, 1H), 3.27 - 3.10 (m, 5H), 3.01 - 2.85 (m, 3H), 2.16 (s, 1H), 2.03 (s, 3H) ppm; 13C NMR (125 MHz, D2O): d = 180.31, 174.84, 155.59, 138.97, 131.08, 130.16, 129.37, 129.16, 127.35, 117.06, 104.60, 101.34, 100.80, 100.00, 81.35, 79.31, 75.24, 74.49, 74.29, 73.52, 72.40, 70.60, 69.76, 68.62, 67.83, 61.08, 60.81, 60.18, 51.07, 40.61, 39.18, 31.89, 22.61 ppm; HRMS: m/r calcd for C37H53N2O18 [M + H]+: 813.3288, found: 813.3289.
[0262] Compound 6: 18 mg (24%) as white solid. ¾ NMR (500 MHz, D20): d = 7.44 - 7.25 (m, 4H), 7.25 - 7.16 (m, 3H), 7.09 - 6.97 (m, 2H), 5.04 - 4.89 (m, 1H), 4.59 (d, J= 8.6 Hz, 1H), 4.41 (dd, J = 7.8, 1.6 Hz, 1H), 4.28 (t, J= 5.9 Hz, 1H), 4.20 (d, = 1.8 Hz, 1H), 4.06 (d, = 3.4 Hz, 1H), 4.01 - 3.91 (m, 1H), 3.83 (d, = 3.6 Hz, 1H), 3.78 - 3.50 (m, 11H), 3.50 - 3.38 (m, 1H), 3.16 (t, J= 7.2 Hz, 2H), 3.07 (t, J= 5.1 Hz, 2H), 2.87 (t, J= 7.2 Hz, 2H), 1.98 (s, 3H), 1.91 (s, 2H) ppm; 13C NMR (125 MHz, D2O): d = 178.68, 177.38, 174.89, 170.28, 155.68, 137.48, 131.17, 130.17, 129.74, 129.63, 128.53, 128.49, 126.76, 126.73, 117.19, 117.16, 104.69, 104.64, 101.97, 100.68, 80.25, 80.22, 80.08,
74.91, 74.61, 74.56, 74.36, 74.06, 72.46, 70.65, 70.12, 68.58, 68.02, 60.96, 60.36, 51.88, 51.33, 42.80,
40.60, 38.18, 31.91, 22.58, 20.94 ppm; HRMS: m/ calcd for C37H53N2O18 [M + H]+: 813.3288, found: 813.3288.
[0263] Compound 7: 15 mg (19%) as white solid. ¾ NMR (500 MHz, D20): d = 7.30 - 7.17 (m, 2H), 7.12 - 7.02 (m, 2H), 5.01 (d, J= 7.5 Hz, 1H), 4.60 (dd, J= 8.6, 2.1 Hz, 1H), 4.45 (d, J= 7.8 Hz, 1H), 4.27 (d, = 2.6 Hz, 1H), 4.16 - 4.09 (m, 1H), 4.01 (ddd, J= 10.5, 8.6, 2.1 Hz, 1H), 3.85 (d, J = 3.5 Hz, 1H), 3.81 - 3.54 (m, 13H), 3.46 (dd, J= 9.9, 7.8 Hz, 1H), 3.19 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.05 (s, 3H), 1.97 (s, 3H) ppm; 13C NMR (125 MHz, D20): d = 174.90, 155.57, 131.20, 130.18, 117.18, 104.75, 102.11, 100.46, 80.47, 78.66, 75.87, 74.89, 74.69, 74.67, 74.42, 73.48, 72.48, 70.67, 69.91, 68.59, 68.06, 60.95, 60.43, 51.32, 40.60, 31.91, 22.55, 17.80 ppm; HRMS: m/z: calcd for C3iH48N2NaOi8 [M + Na]+: 759.2794, found: 759.2795.
[0264] Compound 8: 36 mg (40%) as white solid. ¾ NMR (500 MHz, D20): d = 4.66 (d, J= 8.6 Hz, 1H), 4.38 (d, J= 7.9 Hz, 2H), 4.33 - 4.27 (m, 1H), 4.08 (d, J= 2.0 Hz, 1H), 4.02 (d, J= 3.2 Hz, 1H), 3.95 - 3.82 (m, 4H), 3.80 - 3.75 (m, 1H), 3.74 (dd, J= 10.8, 3.2 Hz, 1H), 3.72 - 3.44 (m, 16H), 3.39 (dd, J= 9.9, 7.8 Hz, 1H), 3.29 (d, J= 6.0 Hz, 2H), 3.18 (dd, J= 9.3, 8.0 Hz, 1H), 3.02 (t, J= 7.0 Hz, 2H), 1.95 (s, 3H), 1.94 - 1.81 (m, 2H), 1.66 - 1.32 (m, 8H), 1.13 - 0.98 (m, 3H), 0.81 (d, J= 11.7 Hz, 2H) ppm; 13C NMR (125 MHz, D20): d = 181.32, 181.18, 174.80, 104.68, 102.94, 101.98, 80.73, 80.41, 80.28, 78.57, 74.84, 74.75, 74.71, 74.47, 74.27, 72.55, 72.39, 70.72, 70.58, 68.50, 67.96, 67.78, 60.87, 60.72, 59.91, 51.30, 40.26, 37.50, 33.17, 33.04, 32.98, 26.60, 26.04, 25.82, 25.76, 22.58 ppm; HRMS: m/z\ calcd for C38H66N2Na023 [M + Na]+: 941.3949, found: 941.3947 [0265] Compound 9: 27 mg (57%) as white solid. ¾ NMR (500 MHz, D20): d = 4.57 (d, J= 8.6 Hz, 1H), 4.43 (dd, .7= 7.9, 2.5 Hz, 2H), 4.39 (d, J= 7.8 Hz, 1H), 4.20 (d, = 2.7 Hz, 1H), 4.12 - 4.05 (m, 2H), 4.02 - 3.88 (m, 3H), 3.84 (d, J= 3.5 Hz, 1H), 3.79 - 3.51 (m, 16H), 3.51 - 3.40 (m, 2H), 3.36 (dd, J= 9.8, 7.8 Hz, 1H), 3.25 (dd, J= 9.2, 8.0 Hz, 1H), 3.09 (t, J= 6.9 Hz, 2H), 2.00 (s, 3H), 1.98 - 1.89 (m, 2H), 1.34 (d, J= 6.7 Hz, 3H) ppm; 13C NMR (125 MHz, D20): d = 180.30, 177.09, 174.84, 104.73, 102.73, 102.10, 102.05, 80.49, 78.99, 78.45, 75.86, 74.89, 74.76, 74.65, 74.31, 74.00, 72.64, 72.48, 70.66, 70.52, 68.58, 68.03, 67.85, 60.95, 60.82, 60.02, 51.29, 37.59, 26.64, 22.55 ppm; HRMS: m/z calcd for C32H59N2Na023 [M + Na]+: 859.3166, found: 859.3165.
[0266] General procedure for the synthesis of cyclohexyl-, phenyllactic acid and lactic acid derivatives 29a-c, 34a-b, and 37a-b: A mixture of compound 23b (for synthesis of 29a-c and 34a-b) or compound 23a (for synthesis of 37a-b) (1 equiv, 0.2N in anhydrous toluene) and Bu2SnO (1.1 equiv.) was heated to reflux under argon atmosphere while stirring. After 2 hours, the clear solution was cooled to RT, and the triflate (1.5 equiv.), anhydrous CsF (2 equiv.), anhydrous DME (equivalent volume to toluene) were added. The suspension was then stirred for 2 hours at RT under argon. The reaction mixture was diluted with EtOAc and washed with water. The organic phase was collected, dried over Na2SC>4, and concentrated. The residue was purified on silica gel MPLC with 10-30% EtOAc/petrol ether to give the product (29a-c, 34a-b, and 37a-b).
[0267] Compound 29a: 416 mg (65%) as colorless oil. ¾ NMR (500 MHz, CDC13): d = 7.39 - 7.17 (m, 35H), 7.12 (d, J= 6.1 Hz, 1H), 7.03 (d, J= 7.4 Hz, 1H), 6.94 (dd, J= 16.2, 7.5 Hz, 5H), 5.03 (d, J = 12.2 Hz, 1H), 4.95 (d, J= 11.2 Hz, 1H), 4.91 - 4.80 (m, 3H), 4.54 (s, 2H), 4.40 (d, J= 11.1 Hz,
1H), 4.33 (dd, J= 7.9, 5.6 Hz, 2H), 4.06 (d, J= 3.5 Hz, 1H), 3.96 - 3.88 (m, 1H), 3.82 (dd, J= 10.0,
5.5 Hz, 1H), 3.75 (dd, J= 9.9, 6.2 Hz, 1H), 3.65 (t, J= 5.7 Hz, 1H), 3.59 (dd, J= 9.2, 3.3 Hz, 1H), 3.48 - 3.29 (m, 3H), 2.84 - 2.63 (m, 5H), 1.77 - 1.57 (m, 13H), 1.38 (dq, J= 9.9, 3.3 Hz, 2H), 1.13 (tt, J= 14.3, 7.6 Hz, 5H), 0.96 - 0.82 (m, 4H) ppm; 13C NMR (125 MHz, CDC13): d = 172.22, 156.12, 138.78, 138.12, 137.90, 136.89, 135.66, 129.81, 128.68, 128.60, 128.55, 128.51, 128.42, 128.25, 128.04, 127.96, 127.82, 127.51, 127.39, 117.28, 102.09, 81.03, 77.94, 76.32, 74.76, 73.80, 73.64, 69.31, 67.41, 66.90, 66.61, 51.06, 40.82, 33.94, 33.71, 33.00, 26.48, 26.22, 26.18 ppm; ESI-MS: m/r calcd for C gHgsNNaOio [M + Na]+: 970.45, found: 970.42.
[0268] Compound 29b: 3.5 g (72%) as colorless oil. ‘HNMR (500 MHz, CDCI3): d = 7.41 - 7.20 (m, 24H), 7.13 (d, J= 7.2 Hz, 1H), 7.02 (d, J= 8.1 Hz, 1H), 6.99 - 6.87 (m, 3H), 5.25 - 5.16 (m, 3H), 5.12 (d, J= 12.0 Hz, 1H), 5.01 (d, J= 11.0 Hz, 1H), 4.92 - 4.85 (m, 1H), 4.76 (d, J= 11.0 Hz, 1H), 4.58 (d, = 11.8 Hz, 1H), 4.53 (d, = 11.9 Hz, 1H), 4.46 - 4.31 (m, 2H), 4.18 (dd, J= 8.6, 4.4 Hz, 1H), 3.94 (dd, J= 9.3, 7.8 Hz, 1H), 3.86 - 3.81 (m, 1H), 3.76 (dd, J= 5.9, 1.8 Hz, 2H), 3.65 (d, J= 6.0 Hz, 1H), 3.49 (s, 1H), 3.39 (qd, J= 15.2, 4.2 Hz, 3H), 2.84 - 2.63 (m, 2H), 1.76 - 1.63 (m, 2H), 1.64 - 1.45 (m, 6H), 1.11 - 0.94 (m, 3H), 0.83 (dqd, J= 15.2, 12.1, 3.2 Hz, 2H) ppm; 13C NMR (125 MHz, CDCI3): d = 174.88, 129.66, 128.70, 128.66, 128.60, 128.58, 128.39, 128.28, 127.83, 127.75, 127.68, 127.57, 117.13, 101.95, 83.37, 77.63, 75.26, 73.73, 73.64, 69.44, 67.20, 66.86, 41.33, 33.75, 33.24, 32.65, 26.31, 25.91 ppm; ESI-MS: m/r calcd for CsgHggNNaOio [M + Na]+: 970.45, found: 970.42.
[0269] Compound 29c: 360 mg (54%) as colorless oil. ‘HNMR (500 MHz, CDCE): d = 7.43 - 7.17 (m, 28H), 7.12 (d, .7= 7.1 Hz, 1H), 7.01 (d, = 8.0 Hz, 1H), 6.95 - 6.85 (m, 4H), 5.17 (s, 2H), 5.04 (d, = 12.2 Hz, 1H), 4.97 - 4.86 (m, 2H), 4.81 (d, = 11.2 Hz, 2H), 4.53 - 4.31 (m, 6H), 3.86 - 3.77 (m, 1H), 3.69 - 3.53 (m, 4H), 3.49 (d, J= 6.2 Hz, 1H), 3.42 (dd, J= 9.3, 3.2 Hz, 2H), 3.39 - 3.31 (m, 1H), 3.10 (dd, = 13.8, 4.1 Hz, 1H), 3.02 - 2.93 (m, 1H), 2.82 - 2.63 (m, 2H) ppm; 13C NMR (125 MHz, CDCE): d = 170.93, 155.96, 138.62, 138.06, 136.91, 135.35, 129.65, 129.33, 128.77, 128.57, 128.55, 128.51, 128.47, 128.41, 128.37, 128.16, 128.12, 127.86, 127.70, 127.66, 127.47, 127.22, 117.18, 101.84, 81.47, 79.15, 74.72, 73.64, 73.32, 68.99, 66.82, 39.49 ppm; ESI-MS: m/ calcd for C59H59NNaOio [M + Na]+: 964.40, found: 964.51.
[0270] Compound 34a: 293 mg (55%) as colorless oil. ‘H NMR (500 MHz, CDC13): d = 7.40 - 7.10 (m, 3 OH), 7.05 - 6.86 (m, 4H), 5.18 (s, 2H), 5.12 (d, = 12.1 Hz, 1H), 5.05 (d, J= 12.1 Hz, 1H), 4.89 - 4.79 (m, 2H), 4.60 - 4.49 (m, 3H), 4.47 - 4.30 (m, 3H), 3.94 (t, J= 2.4 Hz, 1H), 3.86 (dd, J= 9.3, 7.8 Hz, 1H), 3.74 (d, J= 5.7 Hz, 2H), 3.62 (d, J= 5.9 Hz, 1H), 3.48 - 3.31 (m, 3H), 3.08 - 3.02 (m, 3H), 2.83 - 2.64 (m, 2H) ppm; 13C NMR (125 MHz, CDCE): d = 172.89, 155.91, 138.35, 136.39, 135.00, 129.68, 129.56, 128.66, 128.63, 128.57, 128.51, 128.38, 128.32, 128.01, 127.94, 127.86, 127.73, 127.67, 127.63, 126.85, 117.07, 101.90, 81.87, 80.43, 78.69, 74.83, 73.72, 73.71, 69.39, 67.60, 67.16, 50.99, 39.60 ppm; ESI-MS: m/r calcd for CsgHggNNaOio [M + Na]+: 964.40, found: 964.51. [0271] Compound 34b: 414 mg (84%) as colorless oil. ‘HNMR (500 MHz, CDC13): d = 7.40 - 7.26 (m, 19H), 7.14 (s, 1H), 7.07 - 6.90 (m, 4H), 5.24 - 5.17 (m, 3H), 5.11 (d, J = 12.0 Hz, 1H), 4.96 (d, = 10.9 Hz, 1H), 4.86 (t, J= 7.0 Hz, 1H), 4.76 (d, J= 10.8 Hz, 1H), 4.61 - 4.51 (m, 2H), 4.47 - 4.33 (m, 2H), 4.28 (q, J= 6.9 Hz, 1H), 3.97 - 3.89 (m, 2H), 3.82 - 3.71 (m, 2H), 3.64 (d, J= 6.0 Hz, 1H), 3.49 - 3.33 (m, 3H), 2.85 - 2.64 (m, 2H), 1.46 (d, J= 6.9 Hz, 3H) ppm; 13C NMR (125 MHz, CDC13): 5 = 174.20, 156.00, 138.29, 138.13, 128.69, 128.61, 128.57, 128.42, 128.39, 128.18, 127.85, 127.79, 127.74, 127.67, 117.16, 101.90, 82.89, 78.53, 76.76, 76.04, 75.48, 69.45, 67.15 ppm; ESI-MS: m/r calcd for C53H55NNaOio [M + Na]+: 888.37, found: 888.42.
[0272] Compound 37a: 282 mg (56%) as colorless oil. ‘H NMR (500 MHz, CDC13): d = 7.45 - 7.38 (m, 2H), 7.37 - 7.21 (m, 28H), 5.21 (d, J= 12.1 Hz, 1H), 5.13 (d, J= 12.1 Hz, 1H), 5.01 (d, J= 10.6 Hz, 1H), 4.85 - 4.68 (m, 5H), 4.55 (d, J= 12.1 Hz, 1H), 4.48 (d, J= 12.0 Hz, 1H), 4.42 - 4.33 (m, 3H), 4.31 (d, = 7.8 Hz, 1H), 4.16 - 4.08 (m, 2H), 3.99 - 3.91 (m, 2H), 3.79 (dt, = 3.3, 1.6 Hz, 1H), 3.74 (dd, = 10.9, 4.2 Hz, 1H), 3.69 (dd, J= 9.8, 6.9 Hz, 1H), 3.65 - 3.47 (m, 5H), 3.41 - 3.31 (m, 5H), 3.26 (ddd, = 9.8, 4.2, 1.9 Hz, 1H), 3.16 (dd, J= 9.3, 3.2 Hz, 1H), 1.86 (dtd, = 12.3, 6.9, 5.6 Hz, 2H), 1.66 (ddd, J= 13.7, 8.3, 5.3 Hz, 2H), 1.56 - 1.36 (m, 5H), 1.07 - 0.89 (m, 3H), 0.85 - 0.73 (m, 2H) ppm; 13C NMR (125 MHz, CDC13): d = 174.86, 139.03, 138.78, 138.64, 138.52, 138.30, 135.16, 128.69, 128.63, 128.60, 128.45, 128.33, 128.19, 128.07, 127.92, 127.74, 127.66, 127.59, 127.55, 127.50, 127.30, 127.28, 127.17, 103.52, 102.37, 83.82, 82.90, 81.77, 79.13, 77.61, 76.16, 75.47, 75.04, 73.48, 72.99, 68.69, 68.13, 67.17, 66.47, 66.45, 48.35, 41.39, 33.69, 33.23, 32.75, 29.27, 26.29, 25.86, 25.77 ppm; ESI-MS: m/r calcd for C66H77N3NaOi3 [M + Na]+: 1142.53, found: 1142.68. [0273] Compound 37b: 440 mg (93%) as colorless oil. ‘HNMR (500 MHz, CDC13): d = 7.44 - 7.39 (m, 2H), 7.35 - 7.19 (m, 30H), 5.22 (d, J= 12.1 Hz, 1H), 5.13 (d, J= 12.1 Hz, 1H), 5.01 (d, J= 10.7 Hz, 1H), 4.82 (d, J= l LO Hz, 1H), 4.78 - 4.66 (m, 4H), 4.55 (d, J = 12.1 Hz, 1H), 4.47 (d, J= 12.1 Hz, 1H), 4.43 - 4.32 (m, 4H), 4.20 (q, J= 6.9 Hz, 1H), 4.01 - 3.92 (m, 2H), 3.91 - 3.87 (m, 1H), 3.77 (dd, J= 10.9, 4.3 Hz, 1H), 3.73 - 3.65 (m, 2H), 3.65 - 3.54 (m, 3H), 3.50 (dd, J= 9.8, 5.6 Hz, 1H), 3.42 - 3.31 (m, 5H), 3.19 (dt, J= 6.0, 3.3 Hz, 2H), 1.87 (dtd, = 12.2, 7.0, 5.6 Hz, 2H), 1.42 (d, J = 7.0 Hz, 3H) ppm; 13C NMR (125 MHz, CDC13): d = 174.25, 139.03, 138.62, 138.56, 138.44, 138.28, 135.20, 128.70, 128.61, 128.44, 128.36, 128.33, 128.29, 128.25, 128.06, 127.91, 127.81, 127.70, 127.66, 127.61, 127.59, 127.51, 127.49, 127.26, 103.54, 102.42, 83.41, 82.88, 81.78, 79.38, 76.44, 75.81, 75.17, 75.04, 73.54, 73.19, 73.04, 68.72, 68.19, 67.16, 66.84, 66.47, 48.35, 29.28, 19.15 ppm; ESI-MS: m/ calcd for C6oH67N3NaOi3 [M + Na]+: 1060.46, found: 1060.55.
[0274] General procedure of the NIS/TfOH promoted glycosylation for the synthesis of 35a-b and 38a-b: A mixture of glycosyl donor 17 (0.9 equiv.), glycosyl acceptor 34a/b or 37a/b (100-300 mg, 1 equiv.), NIS (1.6 equiv.) and freshly activated 3Ά molecular sieves in anhydrous DCM (2-5 mL) was stirred at RT for 30 minutes. The mixture was then cooled to -20°C, and TfOH (0.1 equiv.) was added. While stirring the reaction mixture was slowly warmed up to -5°C within 2 hours. The reaction mixture was diluted with DCM and washed with sat. NaaSaCh ( q.), sat. NaHCCE (aq.)· The organic phase was collected, concentrated, and the residue was purified on silica gel MPLC with 20-40% EtOAc/petrol ether to give the product (35a-b and 38a-b).
[0275] Compound 35a: 150 mg (29%) as colorless oil. ¾ NMR (500 MHz, CDCh): d = 7.37 - 7.21 (m, 32H), 7.19 - 7.11 (m, 10H), 7.02 - 6.88 (m, 7H), 5.31 - 5.20 (m, 2H), 5.18 (s, 2H), 5.00 (dd, J = 11.8, 9.5 Hz, 2H), 4.96 - 4.89 (m, 2H), 4.80 (q, J= 8.3 Hz, 2H), 4.69 - 4.31 (m, 12H), 4.19 (d, J= 3.2 Hz, 1H), 4.10 (dt, J= 11.2, 5.4 Hz, 2H), 3.89 (ddd, J= 17.9, 8.2, 5.2 Hz, 4H), 3.72 - 3.61 (m, 4H), 3.56 (q, T= 6.3 Hz, 2H), 3.47 - 3.31 (m, 5H), 3.14 (dd, J= 13.9, 4.5 Hz, 1H), 2.99 (dd, J= 14.0, 7.5 Hz, 1H), 2.85 - 2.62 (m, 2H), 2.09 (s, 3H), 2.06 (s, 2H), 2.03 (s, 3H) ppm; 13C NMR (125 MHz, CDCh): d = 172.35, 170.48, 170.07, 169.81, 161.15, 155.82, 138.54, 138.44, 138.34, 137.83, 137.80, 136.04, 135.00, 129.69, 129.48, 129.05, 128.55, 128.52, 128.50, 128.43, 128.43, 128.39, 128.38, 128.32, 128.02, 127.88, 127.82, 127.81, 126.88, 125.31, 117.12, 102.21, 100.82, 99.21, 81.69, 81.31, 79.66, 79.42, 75.15, 74.38, 74.27, 74.00, 73.67, 73.53, 72.75, 72.56, 71.82, 71.07, 70.71, 69.98, 69.11, 68.53, 66.74, 62.11, 55.25, 39.17, 21.22, 20.81, 20.69 ppm; ESI-MS: m/ calcd for CiooHio3Cl3N2Na023 [M + Na]+: 1827.59, found: 1827.70.
[0276] Compound 35b: 200 mg (26%) as colorless oil. ¾ NMR (500 MHz, CDC13): d = 7.41 - 7.21 (m, 38H), 7.20 - 7.10 (m, 2H), 7.04 - 6.87 (m, 4H), 5.29 (t, J= 2.\ Hz, 1H), 5.24 (dd, J= 10.1, 7.9 Hz, 1H), 5.18 (s, 2H), 5.11 - 5.02 (m, 2H), 5.01 - 4.90 (m, 2H), 4.78 (d, J= 8.0 Hz, 1H), 4.66 - 4.24 (m, 12H), 4.17 (d, J= 3.2 Hz, 1H), 4.11 (dd, J= 11.4, 5.8 Hz, 1H), 3.90 (dt, J= 9.7, 6.4 Hz, 3H), 3.82 (dd, J= 9.5, 7.7 Hz, 1H), 3.74 - 3.49 (m, 6H), 3.37 (td, J= 10.4, 3.2 Hz, 4H), 2.84 - 2.63 (m, 2H), 2.09 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.40 (d, J= 6.9 Hz, 3H) ppm; 13C NMR (125 MHz, CDCh): d = 176.61, 173.70, 170.49, 170.06, 169.74, 161.18, 155.86, 138.56, 138.31, 137.84, 137.80, 135.31, 129.04, 128.71, 128.68, 128.57, 128.44, 128.39, 128.35, 128.19, 128.05, 127.88, 127.85, 127.83, 127.82, 127.80, 127.78, 127.75, 127.65, 127.61, 127.56, 127.45, 117.13, 102.16, 100.77, 99.67, 81.63, 79.60, 79.37, 77.28, 77.02, 76.77, 75.33, 74.40, 74.25, 73.89, 73.71, 73.68, 73.52, 72.61, 72.47, 71.77, 71.11, 70.77, 70.07, 68.41, 66.81, 62.20, 55.41, 21.21, 20.79, 20.68, 19.08 ppm; ESI-MS: m/r calcd for CgrHwChN NaC^ [M + Na]+: 1751.56, found: 1751.64.
[0277] Compound 38a: 200 mg (45%) as colorless oil. ¾ NMR (500 MHz, CDCh): d = 7.50 - 7.43 (m, 2H), 7.36 - 7.13 (m, 43H), 5.31 (dd, J= 3.4, 1.1 Hz, 1H), 5.23 (dd, J= 10.2, 7.9 Hz, 1H), 5.11 (d, J= 12.0 Hz, 1H), 5.03 (d, J= 11.9 Hz, 1H), 5.00 - 4.89 (m, 3H), 4.85 - 4.75 (m, 3H), 4.68 (d, J =
10.0 Hz, 1H), 4.63 (d, J= 11.6 Hz, 1H), 4.62 - 4.53 (m, 3H), 4.51 (d, J= 7.9 Hz, 1H), 4.43 (d, J =
12.3 Hz, 1H), 4.41 - 4.36 (m, 3H), 4.35 - 4.27 (m, 3H), 4.21 (d, J= 12.0 Hz, 1H), 4.19 - 4.10 (m,
3H), 4.09 - 4.00 (m, 2H), 4.00 - 3.87 (m, 4H), 3.64 (t, J= 5.7 Hz, 3H), 3.60 - 3.46 (m, 7H), 3.46 - 3.42 (m, 1H), 3.40 - 3.29 (m, 6H), 3.28 - 3.20 (m, 2H), 3.11 (dd, J= 9.6, 3.2 Hz, 1H), 2.21 (s, 3H), 2.01 (s, 3H), 1.91 (s, 3H), 1.85 (dtd, J= 12.1, 6.9, 5.4 Hz, 2H), 1.59 - 1.40 (m, 8H), 1.03 - 0.91 (m, 2H), 0.92 - 0.60 (m, 3H) ppm; 13C NMR (125 MHz, CDCh): d = 173.86, 169.97, 160.92, 138.96, 138.75, 138.54, 138.19, 137.80, 135.26, 129.04, 128.91, 128.79, 128.75, 128.44, 128.37, 128.33, 128.32, 128.26, 128.23, 128.21, 128.19, 128.10, 127.87, 127.85, 127.81, 127.75, 127.58, 127.53, 127.47, 127.36, 127.28, 126.72, 125.30, 103.54, 102.32, 100.68, 99.97, 82.00, 81.90, 79.92, 79.51, 79.31, 75.75, 75.09, 74.73, 74.42, 73.86, 73.52, 73.45, 73.26, 72.94, 72.75, 71.75, 71.04, 70.73, 69.28,
69.28, 68.48, 66.79, 66.42, 62.06, 55.01, 48.32, 33.33, 32.79, 29.25, 26.20, 21.46, 21.06, 20.77 ppm;
ESI-MS: m/z calcd for CiovHmCLNiNaC^ [M + Na]+: 2005.72, found: 2005.93.
[0278] Compound 38b: 137 mg (18%) as colorless oil. ‘HNMR (500 MHz, CDCE): d = 7.49 - 7.46 (m, 2H), 7.41 (d, = 8.2 Hz, 1H), 7.37 - 7.16 (m, 43H), 5.30 (d, J= 3.2 Hz, 1H), 5.24 (dd, = 10.1, 7.9 Hz, 1H), 5.09 (d, J= 12.0 Hz, 1H), 5.00 (d, J= 12.1 Hz, 1H), 4.97 (d, J= 10.1 Hz, 1H), 4.93 (d, = 11.8 Hz, 1H), 4.86 (d, = 8.2 Hz, 1H), 4.84 - 4.74 (m, 3H), 4.70 (d, = 10.0 Hz, 1H), 4.67 - 4.51 (m, 6H), 4.47 - 4.27 (m, 9H), 4.24 - 4.05 (m, 5H), 4.01 (d, J= 3.3 Hz, 1H), 3.92 (ddt, J= 15.6, 11.2, 7.9 Hz, 4H), 3.72 - 3.45 (m, 10H), 3.43 - 3.29 (m, 7H), 3.27 (t, J= 5.7 Hz, 1H), 3.14 (dd, J= 9.5, 3.3 Hz, 1H), 2.21 (s, 3H), 2.01 (s, 3H), 1.93 (s, 3H), 1.90 - 1.81 (m, 3H), 1.35 (d, J= 6.9 Hz, 3H) ppm; 13C NMR (125 MHz, CDCE): d = 173.65, 170.51, 169.98, 169.93, 161.04, 138.93, 138.69, 138.64, 138.50, 138.19, 137.78, 137.76, 135.26, 128.41, 128.40, 128.36, 128.33, 128.29, 128.28, 128.25, 128.23, 128.19, 128.18, 127.86, 127.82, 127.53, 127.47, 103.55, 102.26, 100.60, 100.39, 82.78, 81.77, 81.54, 80.03, 79.54, 77.28, 77.03, 76.77, 75.93, 75.84, 75.74, 75.14, 75.10, 74.46, 73.97, 73.67, 73.52,
73.45, 73.42, 73.27, 72.67, 72.59, 71.77, 71.01, 70.49, 69.25, 69.16, 68.49, 68.09, 66.94, 66.45, 61.99,
54.89, 48.33, 29.27, 21.08, 20.74, 20.72, 18.97 pm; ESI-MS: m/z: calcd for CioiHmCWN NaOai [M + Na]+: 1923.64, found: 1923.73.
[0279] General procedure oftrans-esterification/de-acetylation for the synthesis of 36a-b and 39a-b: To a mixture of 35a/b or 38a/b (100-200 mg, 1 equiv.) and anhydrous MeOH (2-4 mL), 25% w/w NaOMe/MeOH (0.1 equiv.) was added at RT. The reaction mixture was stirred at RT and the progress of reaction was monitored by TLC. After reaction completion, the mixture was neutralized with IR- 120 (H+) resin to pH 7 and then filtered. The filtrate was concentrated under vacuum and the residue was used directly in next hydrogenation step.
[0280] General procedure for the synthesis of mercaptobutanamide-fimctionalized GM1 mimetics (40-48): To a solution of GM1 mimetic (1-9) (4-20 mg, 1 equiv.) in dry methanol (2-10 mL), triethylamine (10 equiv.) and g-thiobutyrolactone (10 equiv.) were added. The reaction mixture was stirred under argon atmosphere at RT overnight. The reaction mixture was concentrated under vacuum. The residue was dissolved with DI water and extracted with EtOAc three times. The aqueous phase was then lyophilized to give the product (40-48) as white solid, which was used directly in next step.
[0281] General procedure for the synthesis of chloroacetylated poly-Z -lysine 54-58, 86-88: Poly-L- lysine (hydrobromide or trifluoroacetic acid) 49-53, 83-85 (100 mg, 0.48 mmol of lysine units) was suspended in a mixture of DMF (2 mL) and 2,6-lutidine (0.5 mL). The mixture was cooled in ice- water bath, and then chloroacetic anhydride (244 mg in 0.5 mL of DMF) was added over 10 min. Poly-l-lysine was gradually dissolved during the addition of the chloroacetic anhydride solution. The reaction mixture was then stirred at 4 °C for 16 hours. The product was precipitated by drop-wise addition of the reaction mixture to vigorously stirring solvent mixture (ethanol/diethyl ether 1 : 1, 40 mL). The mixture was centrifuged at 1000 rpm, 4-10 °C, for 2 min. The solvent was decanted, and the residue was re-suspended in ethanol/ether (1:1, 30 mL). The centrifugation and re-suspension was repeated three times. The product precipitates were collected and dried under vacuum overnight to give the products 54-58, 86-88 as white solids (77-98%). 1HNMR data were identical to the previous report. [Ref: G. Thoma et al., J. Am. Chem. Soc. 1999, 121: 5919-5929]
[0282] General procedure for the synthesis of gly copolymers 59-82, 89-97: To a stirring solution of chloroacetylated poly-Z -lysine 54-58, 86-88 (1 equiv.) in DMF (0.04 M), a solution of GM1 mimetic 40-48 (0.2-0.7 equiv. depending on target epitope loading) in water (0.1 M) was added. N,N- Diisopropylethylamine (2 equiv.) and l,8-diazabicyclo[5.4.0]undec-7-ene (1 equiv.) were added to the above mixture. The reaction mixture was then stirred at RT under argon atmosphere for 45 min. 1- Thioglycerol (3 equiv.) and triethylamine (3 equiv.) were added and the mixture was stirred for additional 16 hours. The reaction mixture was dropped into a stirring solution of diethyl ether/ethanol (1:1; 3ml), leading to the precipitation of the polymer. The precipitates were collected by centrifugation, washed with ethanol (3 mL), and then dissolved with 10 mL of water. The crude product solution was adjusted to pH 9-10 by addition of 1M NaOH. Further purification was achieved by means of ultrafiltration. Ultracentrifugation was performed using Sartorius Stedim Vivaspin tubes (volume 15 mL, MWCO 10-100 kDa depending on polymer length). The ultracentrifugation was repeated four times (from 10 mL to 1 mL), on each occasion the volume was filled up with DI water. Lyophilization gave the GM1 mimetic-polymer conjugates 59-82 & 89-97 (57-99%) as white solids. According to 'H-N R. the products contained approximately 12-37% monomer carbohydrate units linked to the polymer. Representative epitope/mimetic loadings were listed below:
[0283] Polymer 59: natGMl epitope (l)-polymer 56 conjugate, monomer loading: 26%.
[0284] Polymer 60: GM1 mimetic (2)-polymer 56 conjugate, monomer loading: 30%.
[0285] Polymer 61: GM1 mimetic (3)-polymer 56 conjugate, monomer loading: 24%.
[0286] Polymer 62: GM1 mimetic (4)-polymer 56 conjugate, monomer loading: 25%.
[0287] Polymer 63: GM1 mimetic (5)-polymer 56 conjugate, monomer loading: 25%.
[0288] Polymer 64: GM1 mimetic (6)-polymer 56 conjugate, monomer loading: 26%.
[0289] Polymer 65: GM1 mimetic (7)-polymer 56 conjugate, monomer loading: 26%.
[0290] Polymer 66: GM1 mimetic (8)-polymer 56 conjugate, monomer loading: 35%.
[0291] Polymer 67: GM1 mimetic (9)-polymer 56 conjugate, monomer loading: 30%.
[0292] Polymer 68: GM1 mimetics (2/4, l:2)-polymer 56 conjugate, monomer loading: 30%.
[0293] Polymer 69: GM1 mimetic (2)-polymer 54 conjugate, monomer loading: 14%. [0294] Polymer 70: GM1 mimetic (2)-polymer 54 conjugate, monomer loading: 30%.
[0295] Polymer 71: GM1 mimetic (2)-polymer 54 conjugate, monomer loading: 37%.
[0296] Polymer 72: GM1 mimetic (2)-polymer 55 conjugate, monomer loading: 14%.
[0297] Polymer 73: GM1 mimetic (2)-polymer 55 conjugate, monomer loading: 29%.
[0298] Polymer 74: GM1 mimetic (2)-polymer 55 conjugate, monomer loading: 37%.
[0299] Polymer 75: GM1 mimetic (2)-polymer 56 conjugate, monomer loading: 12%.
[0300] Polymer 76: GM1 mimetic (2)-polymer 56 conjugate, monomer loading: 37%.
[0301] Polymer 77: GM1 mimetic (2)-polymer 57 conjugate, monomer loading: 14%.
[0302] Polymer 78: GM1 mimetic (2)-polymer 57 conjugate, monomer loading: 28%.
[0303] Polymer 79: GM1 mimetic (2)-polymer 57 conjugate, monomer loading: 36%.
[0304] Polymer 80: GM1 mimetic (2)-polymer 58 conjugate, monomer loading: 12%.
[0305] Polymer 81: GM1 mimetic (2)-polymer 58 conjugate, monomer loading: 28%.
[0306] Polymer 82: GM1 mimetic (2)-polymer 58 conjugate, monomer loading: 36%.
[0307] Polymer 89: GM1 mimetic (2)-polymer 86 conjugate, monomer loading: 13%.
[0308] Polymer 90: GM1 mimetic (2)-polymer 86 conjugate, monomer loading: 29%.
[0309] Polymer 91: GM1 mimetic (2)-polymer 86 conjugate, monomer loading: 37%.
[0310] Polymer 92: GM1 mimetic (2)-polymer 87 conjugate, monomer loading: 14%.
[0311] Polymer 93: GM1 mimetic (2)-polymer 87 conjugate, monomer loading: 29%.
[0312] Polymer 94: GM1 mimetic (2)-polymer 87 conjugate, monomer loading: 36%.
[0313] Polymer 95: GM1 mimetic (2)-polymer 88 conjugate, monomer loading: 14%.
[0314] Polymer 96: GM1 mimetic (2)-polymer 88 conjugate, monomer loading: 29%.
[0315] Polymer 97: GM1 mimetic (2)-polymer 88 conjugate, monomer loading: 36%.
[0316] Exemplary procedure for the synthesis of GM1 mimetic (2)-fimctionalized Sepharose beads 98: Epoxy-activated Sepharose 6B (0.5 g, 1.75 ml final volume, 70 mhioΐ active groups) was suspended in 100 ml distilled water for lh in 10x10 ml aliquots and filtered. GM1 mimetic (2) (34.2 mg, 0.035 mmol, 0.50 eq) was dissolved in 1.75 ml carbonate buffer (0.1 M carbonate buffer + 0.15 M NaCl) and added to the Epoxy-activated Sepharose 6B in a closed vial. The mixture was shaken for 24 h at RT. Then, 2-aminoethanethiol (27 mg, 0.35 mmol, 5.00 eq, 1M in buffer) was added for capping and the mixture was shaken for another 16h at RT. For work-up, the mixture was filtered and washed 3 times with alternatively 0.1 M NaOAc pH 4 containing 0.5 M NaCl followed by 0.1 M Tris- HC1 pH 8 containing 0.5 M NaCl, and dried on the glass frit.
[0317] Alternatively, NHS-activated Sepharose 4 Fast Flow was used in the preparation of GM1 mimetic (2)-functionalized Sepharose beads 99: In deviation from the above procedure, thioglycerol (38 mg, 0.35 mmol, 5.00 eq, 1M in buffer) was used for capping. 6.2. Example 2. Assessment of Activity of Compounds.
6.2.1. Patient Sera
[0318] Sera from GBS, MMN and control neuropathy patients or healthy individuals were investigated. They were tested for anti-GMl IgG and IgM antibodies by ELISA. Serum anti-GMl antibody titers were determined by an ELISA assay from Biihlmann Laboratories (Schonenbuch, Switzerland). Sera were either obtained from University Medical Center Utrecht (Utrecht, Netherlands) or the immunology laboratory of the University Hospital Marseille (Marseille, Prance). Sera from healthy individuals (without neuropathy) or from control neuropathy patients both negative for anti-GMl reactivity served as control and were obtained from the blood bank in Basel (Blutspendezentrum SRK beider Basel, Basel, Switzerland). All participants signed an informed consent.
6.2.2. Direct binding and competitive binding Assay
[0319] The synthesized carbohydrate polymers 60-97 (glycomimetics of the GM1 epitope), were tested and compared to the synthesized carbohydrate polymer 59 (natural GM1 epitope) for direct binding activity of anti-GMl IgM and IgG and in a competitive GM1 ELISA (Biihlmann Laboratories, Schonenbuch, Switzerland).
[0320] Lor the direct binding assay, Maxisorp plates (Thermo Scientific, Switzerland) were coated with 100 pL/well of 2 pg/ml the polymer 60 (in phosphate buffered saline solution (PBS), Lonza, Switzerland) and incubated overnight at 4-8°C. The plates were washed four times with 300 pL/well Wash Buffer (0.05% Tween20 in PBS). To block unspecific binding the plates were afterwards incubated with 100 pL of blocking buffer (5% Bovine serum albumin (BSA) in PBS) for two hours at room temperature. The blocking buffer was discarded without a washing step prior adding the 100 pL human serum diluted 1:50 in incubation buffer (2% BSA, 0.05% Tween20 in PBS) for two hours at 4- 8°C. After the incubation step, the plates were washed four times with 300 pL/well Wash Buffer and incubated for two hours at 4-8°C with 100 pL/we 11 of undiluted horseradish peroxidase-labeled anti human IgG or IgM. The plate was washed four times with 300 pL/well Wash Buffer before 100 pL/we 11 undiluted tetramethylbenzidine (TMB, citrate buffer with hydrogen peroxide) substrate (Thermofisher, Switzerland) was added and incubated for 30 minutes at room temperature on a plate rotator (600 revolutions per minute, rpm). Before the absorbance was measured, 100 pL/well stop solution (0.25 M sulfuric acid) was added. The degree of colorimetric reaction was determined by absorption measurement at 450 nm with a microplate reader (Synergy HI, Microplate reader,
BioTek).
[0321] Lor the competitive ELISA, the synthesized carbohydrate polymers 60-68 were tested in the GM1 ELISA (Biihlmann Laboratories, Schonenbuch, Switzerland) for their inhibitory activity (inhibition of patients’ serum anti-GMl IgG and IgM antibodies to the GM1 ganglioside). The 96 well microtiter plates coated with purified GM1 ganglioside were washed two times with Washing Buffer (300 mΐ/well) before adding the 50 pi carbohydrate polymers dissolved in PBS and 50 mΐ/well diluted patient sera (1:25 dilution in PBS). The final volume was 100 mΐ/well of the mixture per well which was incubated for two hours at 4-8°C. The plates were washed four times with wash buffer (300 mΐ/well) before either the anti -human IgM antibody-horseradish peroxidase conjugate or the anti human IgG antibody-horseradish peroxidase conjugate was added (100 mΐ/well). The plate was incubated for two hours at 4-8°C. After washing the wells (4 x 300 mΐ/well), the substrate solution TMB was added (100 mΐ/well) and the plate incubated for further 30 minutes at 600 rpm and room temperature, protected from light. Finally, a stop solution (0.25 M sulfuric acid) was added (100 mΐ/well) and the degree of colorimetric reaction was determined by absorption measurement at 450 nm with a microplate reader (Synergy HI, Microplate reader, BioTek).
[0322] To determine the in vitro IC50 of polymer 60, 69-97, the assay was performed with co incubation of polymer (50 mΐ/well) and anti-GMl IgG+ GBS patient sera.
[0323] To assess the anti-GMl binding of GM1 mimetic (2)-fimctionalized Sepharose beads 98 and 99 on a column, the following procedure was used: A layer of cotton was added to the bottom of a 2 ml plastic syringe to form as basic column setup. The column was rinsed with PBS and 500 mΐ of GM1 mimetic (2)-functionalized Sepharose beads 98 or 99 (settled bead volume in PBS), were added on top of the cotton layer. The column was rinsed with 2 ml of PBS, then various volumes of patient serum (100-500 mΐ) were added per column and allowed to flow through the column by gravity. The eluate was collected, a sample was taken from it and diluted 1:50 for analysis by the GM1 ELISA (Biihlmann Laboratories, Schonenbuch, Switzerland) as described above. In case of multiple flows through the column, the eluate was collected and added on top of the column again to allow another flow through the column, this was done up to 3x in selected experiments.
6.2.3. Animal model of acute motor axonal neuropathy (AMAN)
[0324] For the anti-GMl IgG antibody mediated AMAN mouse model, GalNAcT-/--Tg(neuronal) transgenic on a C57B1/6 background were used (see e.g., McGonigal, R., etal., Clq-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy. Acta Neuropathol Commun, 2016. 4: p. 23). All mice were four weeks old (12-15 g), had unlimited access to food and water, and were housed with a light/dark cycle of 12 h/12 h at constant temperature of 22 °C. Mice of either sex were sacrificed by CO2 inhalation. All experiments using mice were performed in accordance with a licence approved and granted by the United Kingdom Home Office.
[0325] Mice were injected intraperitoneally (i.p.) with 4 mg anti-GMl ganglioside IgG. After 2 h, mice were injected intravenously (i.v.) into the tail vein with 2 mg of the glycopolymer 60 and after a further 30 min interval, 0.5 ml 100 % normal human serum (NHS) was delivered i.p. At 6 h, mice were asphyxiated with a rising concentration of CO2. Blood, diaphragm, and soleus muscles were collected for serum and immunohistochemical analysis.
6.2.4. Ex vivo immunostaining of target tissue
[0326] The immunostaining was performed according to the publication by McGonical et al. 2016 (McGonigal, R., et al. , Clq-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy. Acta Neuropathol Commun, 2016. 4: p. 23). Diaphragms were removed upon termination of the experiment, snap frozen, and stored at -70°C. Tissue was mounted in mounting medium and longitudinal cryosections collected at 8-15 pm on to coated slides. Sections were stored at -20 °C until use. Immunostaining for anti-GMl IgG antibody binding and complement deposits at the nerve terminal was performed as described elsewhere (Halstead, S.K., et al, Eculizumab prevents anti-ganglioside antibody-mediated neuropathy in a murine model. Brain, 2008. 13 l(Pt 5): p. 1197-208). The nerve terminals, were identified by Alexa Fluor 555 conjugated a-bungarotoxin (a-BTx, 1.3 pg/ml, Molecular Probes). FITC conjugated goat anti-mouse IgG2a (1:300, Southern biotech). All detection antibodies were diluted in PBS. For the assessment of anti-GMl IgG antibody binding only, tissue was washed and fixed with 4% paraformaldehyde in PBS immediately after anti-GMl IgG3 (DG2) incubation. Application of 0.1 M glycine for 10 min was performed to quench unreactive aldehyde groups. Tissue was then incubated overnight at 4°C with anti-mouse IgG/M-FITC (1:300) in PBS. Tissue was rinsed in PBS and mounted in Citifluor mounting medium (Citifluor Products, UK).
[0327] Image capture and analysis. Digital images were captured using both a Zeiss Pascal confocal laser scanning microscope and a Zeiss Axio Imager Z 1 with ApoTome attachment. For quantitative analysis of IgG and C3c staining was performed in triplicate for each marker, and quantified as previously described. All studies were observer blinded and statistically analysed using GraphPad Prism 7 software.
6.2.5. Inhibitory activity against anti-GMl antibodies from patient sera.
[0328] The invented glycopolymers are based on a biodegradable poly-F-lysine backbone and carbohydrate epitopes that imitate the natural glycoepitope of the GMl-ganglioside. The polymers 59- 97 are designed for a therapeutic application in patients, where pathogenic anti-GMl antibodies could be selectively neutralized and removed. These anti-GMl IgG and IgM isotype autoantibodies are involved in autoimmune neurological diseases; their binding to GM1 in the peripheral nervous system triggers demyelination and neurodegeneration (see e.g., Willison, H.J. and N. Yuki, Peripheral neuropathies and anti-glycolipid antibodies. Brain, 2002. 125(Pt 12): p. 2591-625). For the biological evaluation of the prepared glycopolymers, a collection of patient sera samples and an AMAN mouse model was used. Initially, the sera samples were tested positive for anti-GMl IgM or IgG antibodies. The synthetic glycopolymers 60-68 were tested for the inhibitory activity to block the binding of anti- GM1 antibodies to GM1 ganglioside. The different inhibitory activities were compared to the polymer carrying the natural GM1 glycoepitope 59 at two to four different concentrations in the competitive binding ELISA assay with a selection of patient sera.
[0329] FIG. 3A shows inhibition of binding of selected MMN patients’ sera anti-GMl IgM to GM1 ganglioside by exemplary polymeric compounds 59-68. FIG. 3B is a plot of inhibitory activity of polymeric compounds 59, 60, and 62 to block the binding of MMN patients’ sera anti-GMl IgM to GM1 ganglioside (n = 23 MMN patients, ***p> 0.001, **** p>0.00001).
[0330] The inhibitory activity obtained during the biological characterization showed the different neutralization effects of the gly copolymers for anti-GMl IgM antibodies from different patients (FIG. 3A). This is probably due to interindividual differences of antibody characteristics (isotype, affinity, specificity, serum concentration, monoclonal/polyclonal, etc.) between the different patients.
However, the inhibitory effect of the glycopolymers 59, 60, and 62 was best throughout the tested patient cohort (FIG. 3B), with polymer 60 showing most pronounced inhibition. Furthermore, the data indicates that GM1 epitope mimics including a sialic acid group can provide potent inhibitory activity to neutralize anti-GMl IgM antibodies efficiently in serum of a large patient cohort. The IC50 values of polymers 60, 69-97 were determined with anti-GMl antibody positive GBS patients’ sera. The glycopolymer 60 exhibited an inhibitory activity in the picomolar to nanomolar range (Table 1). [0331] FIG. 3C-3D shows the depletion of anti-GMl IgG (FIG. 3C) and anti-GMl IgM (FIG. 3D) antibodies from patient serum. 100-500 pi of patient serum were allowed to flow through (lx, top or 3x, bottom) a column containing 500 mΐ of GM1 mimetic (2) -functionalized Sepharose beads 98 (FIG. 3C) and 99 (FIG. 3D) by gravitational force. The eluate was collected for analysis of the amount of anti-GMl IgG or IgM antibodies remaining in the eluate after flowing through the column compared to the untreated serum before filtration. In FIG. 3D graph, the anti-GMl IgM absorbance of a healthy donor control sample is shown as a control (background).
[0332] Table 1. Inhibition of the binding of GBS patients’ anti-GMl IgG and IgM to the GM1 ganglioside by polymer 60.
Figure imgf000070_0001
[0333] Since IgM binding can be temperature dependent, we investigated the inhibitory activity at three different temperatures (4°C, 25°C, 37°C). FIG. 4 shows the effects of temperature on the inhibitory activity of exemplary polymeric compounds. Temperature dependent inhibition of anti- GM1 IgG to the GM1 ganglioside by polymer 62 and temperature independent inhibition by polymers 60 and 68 (***p> 0.001).
[0334] We observed that polymer 60 was highly active independent of the temperature during the incubation (FIG. 4), whereas polymer 62 exhibited a decrease of the inhibitory activity at 37°C.
6.2.6. Evaluation of polymer characteristics on inhibitory activity.
[0335] In addition, modifications of polymers 54-58 and 86-88 with different GM1 mimetic (2) epitope loading were tested in vitro to evaluate the impact of polymer length and epitope loading (69- 97) on inhibitory activity against a monoclonal anti-GMl IgG antibody in a competitive ELISA (Table 2). The polymer 60 exhibited a higher inhibitory activity with patients’ sera, in contrast to the inhibition of the monoclonal anti-GMl IgG.
[0336] Table 2. Inhibition of the binding of a monoclonal anti-GMl IgG to the GM1 ganglioside by polymer 69-97.
Figure imgf000071_0001
Figure imgf000072_0001
means no inhibition was detected in assay
6.2.7. Activity of compounds in the mouse model of acute motor axonal neuropathy (AMAN).
[0337] The polymer 60 and related polymers 61-97 are designed for a therapeutic application in peripheral neuropathies associated with anti-GMl IgG and IgM autoantibodies (e.g. AMAN,
AMSAN, and MMN). We evaluated whether pathogenic anti-GMl antibodies could be selectively neutralized and removed by polymer 60 in an established animal model for AMAN (McGonigal, R., et al. , Clq-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy. Acta Neuropathol Commun, 2016. 4: p. 23).
[0338] FIG. 5 shows ex vivo inhibition of anti-GMl IgG binding and complement deposition to murine nerve terminals by polymer 60 (***p> 0.001).
[0339] Polymer 60 inhibited the binding of the mouse monoclonal anti-GMl IgG to the GM1 epitope on murine diaphragm tissue at ex vivo (FIG. 5). The therapeutic utility of polymer 60 is further supported by inhibition of human complement deposition (C3c) at the target tissue. Given that anti- GMl antibody-mediated complement activation is a key driver for axonal damage in anti-GMl antibody-related peripheral neuropathies.
[0340] This finding was confirmed in the in vivo nerve injury model. FIG. 6A-6C shows inhibition of anti-GMl IgG binding to phrenic nerves by exemplary polymeric compound 60 in the in vivo nerve injury mouse model. The i.v. injection of polymer 60 did reduce the binding of the anti-GMl IgG antibody (DG2) to the background signal (auto-fluorescence) in the target tissue, i.e., phrenic nerves in the diaphragm tissue (FIG. 6B, 6C). a-Bungarotoxin (Btx) was used to stain the nicotinic acetylcholine receptors (nAChRs) and confirm the tissue samples were isolated and analyzed correctly (FIG. 6A, 6B). We confirmed the neutralization and removal of the anti-GMl antibody by polymeric compound 60 in the serum by ELISA. The data from the in vivo nerve injury model demonstrates that the neutralization and removal of anti-GMl antibodies from the circulation is a suited treatment approach to minimize autoantibody-mediated nerve damage. Furthermore, it indicates that the polymer 60 is acting upstream of the complement cascade activation and therefore, inhibits complement-meditated axonal damage as well. 6.3. Example 3. Assessment of efficacy of an exemplary polymeric compound (polymer 60) in binding and sequestering anti-GMl antibodies to prevent autoreactive antibody binding.
6.3.1. Methods
[0341] The enzyme required to produce GM1 gangliosides, GalNAcT, is driven by the Thyl promoter for neuronal membranes. Mice were bred with GM1 restricted neuronally to study selective GM1 binding along the axon (see e.g., Yao, D., et al. Journal of Neuroscience 2014, 34(3), 880-891). [0342] Diaphragm sections and triangularis stemi (TS) nerve-muscle preparations from neuronal GM1 -enriched mice were used to assess binding in vitro and ex vivo, respectively. FIG. 7A depicts sections of murine diaphragm used for in vitro investigation. FIG. 7B illustrates triangularis stemi muscle, dissected out of mouse rib cage, used for ex vivo investigation (created using BioRender). [0343] In vitro preparations were incubated with anti-GMl antibody (DG2) and exemplary polymeric compound (polymer 60; “mimetic 1” = GM1 mimetic (2)) or control polymeric compound (control mimetic) to assess dose dependence of the mimetic. Ex vivo preparations were incubated in Ringer’s solution with both DG2 and the GM1 gly copolymer or the control gly copolymer. After the incubation period, antibody and polymeric compound were removed by rinsing, following this the TS muscle or diaphragm was stained with bungarotoxin to visualize the nerve-terminal and an anti-mouse IgG3 antibody to measure DG2 binding. Images were captured using a Zeiss LSM 880 Confocal Microscope.
6.3.2. Results
[0344] FIG. 8A-8B. exemplary polymeric compound (polymer 60; “mimetic 1” = GM1 mimetic (2)) fully sequesters anti-GMl antibody DG2 in vitro. FIG. 8A shows a graph of in vitro dose response for binding and sequestering anti-GMl antibodies by the exemplary polymeric compound as compared to a control polymeric compound (control mimetic) that does not bind anti-GMl antibody. FIG. 8B shows the related images of diaphragm tissue from neuronal GM1 -enriched mice. Btx=nicotinic acetylcholine receptor; DG2=anti-GMl antibody. Scale Bar = 50 pm.***p < 0.001, **p < 0.01, one way ANOVA.
[0345] FIG. 9A-B show that exemplary polymeric compound (polymer 60; “mimetic 1” = GM1 mimetic (2)) reduces anti-GMl antibody binding ex vivo. FIG. 9A shows a graph of DG2 antibody staining observed in the tissue for exemplary polymeric compound as compared to control polymeric compound (control mimetic) that does not bind anti-GMl antibody. FIG. 9B shows images of triangularis stemi nerve-muscle tissue preparations from the neuronal GM1 -enriched mice stained with bungarotoxin to visualize the nerve-terminal and an anti-mouse IgG3 antibody to measure DG2 antibody. Btx=nicotinic acetylcholine receptor; DG2=anti-GMl antibody. Scale Bar = 20 pm;****p < 0.0001, unpaired two-tailed student t-test. [0346] The exemplary polymeric compound (polymer 60; “mimetic 1” = GM1 mimetic (2)) successfully sequesters anti-GMl antibody in vitro at concentrations as low as 0.5 pg/mL.
[0347] The exemplary polymeric compound (polymer 60; “mimetic 1” = GM1 mimetic (2))binds and sequesters anti-GMl antibody ex vivo at a concentration of 50 pg/mL.
[0348] These results indicate the anti-GMl antibody-binding compounds and conjugates of this disclosure are active in vivo and could be useful in the treatment of indications involving anti-GMl antibodies such as AMAN.
7. EQUIVALENTS AND INCORPORATION BY REFERENCE
[0349] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
[0350] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

WHAT IS CLAIMED IS:
1. An anti-GMl antibody-binding compound of formula (I) comprising a plurality of glycans linked to a moiety of interest:
Figure imgf000075_0001
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group;
Z1 is -0-, -S-, -NR2- or -C(R2)2-, wherein each R2 is independently selected from H, (C1-C4)- alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5;
Ar is optionally substituted aryl or optionally substituted heteroaryl;
L1 is a linker; m is at least 2; and Y is the moiety of interest.
2. The compound of claim 1, wherein the compound is of formula (II):
Figure imgf000075_0002
or a pharmaceutically acceptable salt thereof, wherein: q is 0 to 4; and each R11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
3. The compound of claim 1 or 2, wherein: R1 is selected from:
Figure imgf000076_0001
R21 is optionally substituted (Ci-C3)-alkyl; and
R22 and R23 are independently selected from H, optionally substituted (Ci-C3)-alkyl, optionally substituted aryl-(Ci-C3)-alkylene-, optionally substituted heteroaryl-(Ci-C3)-alkylene-, optionally substituted cycloalkyl-(Ci-C3)-alkylene-, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl.
4. The compound of claim 3, wherein R1 is
Figure imgf000076_0002
or a pharmaceutically acceptable salt thereof.
5. The compound of claim 3, wherein R1 is
Figure imgf000076_0003
or a pharmaceutically acceptable salt thereof.
6 The compound of claim 3, wherein R1 is a substituted carboxymethyl group selected from:
Figure imgf000076_0004
or a pharmaceutically acceptable salt thereof.
7. The compound of claim 6, wherein R1 is selected from:
Figure imgf000077_0001
or a pharmaceutically acceptable salt thereof.
8. The compound of any one of claims 1 to 7, wherein Z1 is -0-.
9. The compound of any one of claims 1 to 7, wherein Z1 is -S-.
10. The compound of any one of claims 1 to 7, wherein Z1 is -NR2-.
11. The compound of any one of claims 1 to 7, wherein Z1 is -NH-.
12. The compound of any one of claims 1 to 7, wherein Z1 is -C(R2)2-.
13. The compound of any one of claims 1 to 7, wherein Z1 is -CH2-.
14. The compound of any one of claims 1 to 13, wherein L1 is linear linker comprising one or more linking moieties independently selected from -Ci-6-alkylene-, -NHCO-Ci-6-alkylene-, -CONH- Ci-6-alkylene-, -NH Ci-6-alkylene-, -NHCONH-Ci-6-alkylene-, - NHCSNH-Ci-6-alkylene-, -C1-6- alkylene-NHCO, -Ci-6-alkylene-CONH-, -Ci-6-alkylene-NH-, -Ci-6-alkylene-NHCONH-, -C1-6- alkylene-NHC SNH-, -0(CH2)p- -(OCH2CH2)p-, -NHCO-, -CONH-, -NHS02- -S02NH- -CO-, — S02— , -0-, — S — , pyrrolidine-2,5 -dione, -NH-, and -NMe-, wherein p is 1 to 10.
15. The compound of claim 14, wherein L1 is -(CH2)2NH(C=0)(CH2)3S-CH2-(C=0)- connecting Ar to an amino-containing group of Y.
16. The compound of any one of claims 1 to 15, wherein Y is a solid support (e.g., bead, nanoparticle, planar support, 96-well plate, etc.).
17. The compound of any one of claims 1 to 16, wherein Y comprises a polymer.
18. The compound of claim 16 or 17, wherein Y is a bead.
19. The compound of 18, wherein Y is an agarose, a sepharose, a dextran, a cellulose, chitin, chitosan, an organic or inorganic porous material, a magnetic bead, or a micro bead.
20. The compound of any one of claims 1 to 17, wherein Y is a polypeptide or a polysaccharide.
21. The compound of claim 20, wherein Y comprises an a-amino acid polymer backbone having a mean length of 10 to 5000 amino acid residues (e.g., 20 to 2000, 30 to 1000, or 50 to 800 amino acid residues).
22. The compound of claim 21, wherein the a-amino acid polymer backbone has a mean length of 50 to 800 amino acid residues (e.g., 100 to 600 amino acid residues, such as 100, 150, 200, 300, 400, or 600).
23. The compound of any one of claims 19 to 21, wherein the a-amino acid polymer backbone has a mean length of at least 200 amino acid residues.
24. The compound of claim 21, wherein the a-amino acid polymer backbone has a mean length of 360 to 440 amino acid residues (e.g., 380 to 420, or 390 to 410 amino acid residues, such as about 400 amino acid residues).
25. The compound of any one of claims 20 to 24, wherein Y is an a-amino acid polymer consisting essentially of a-amino acid residues selected from lysine, ornithine, glutamic acid and aspartic acid.
26. The compound of claim 25, wherein the a-amino acid polymer is poly-lysine.
27. The compound of claim 26, wherein the a-amino acid polymer is poly-Z-lysine.
28. The compound of claim 26, wherein the a-amino acid polymer is poly-D-lysine.
29. The compound of claim 25, wherein the a-amino acid polymer is poly-glutamic acid.
30. The compound of any one of claims 20 to 29, wherein 10% to 50% of the residues of the a- amino acid polymer are linked to the glycans of formula (I).
31. The compound of claim 30, wherein 10% to 40% of the residues of the a-amino acid polymer are linked to the glycans of formula (I).
32. The compound of claim 31, wherein 20% to 40% of the residues of the a-amino acid polymer are linked to the glycans of formula (I).
33. The compound of claim 32, wherein 25% to 40% of the residues of the a-amino acid polymer are linked to the glycans of formula (I).
34. The compound of any one of claims 30 to 33, wherein the loading of amino acids residues of the a-amino acid polymer with the glycans of formula (I) is determined by NMR or quantitative NMR.
35. The compound of any one of claims 30 to 34, wherein the a-amino acid polymer is poly lysine and the remaining lysine side chains in the poly-lysine polymer are capped with a water solubilizing substituent.
36. The compound of claim 35, wherein the remaining lysine side chains in the poly-lysine polymer are capped with 2,3-dihydroxypropylthioacetyl.
37. The compound of any one of claims 1 to 36, wherein R2 is selected from:
Figure imgf000079_0001
38. The compound of claim 37, wherein the multitude of glycans of formula (I) have the structure:
Figure imgf000079_0002
39. The compound of claim 37, wherein the multitude of glycans of formula (I) have the structure:
Figure imgf000080_0001
40. The compound of any one of claims 37 to 39, wherein:
L1 is -(CH2)2NH(C=0)(CH2)3S-CH2-(C=0)-;
Y is poly-lysine polymer backbone having a mean length of 360 to 440 amino acid residues; 25% to 40% of the lysine side chains in the a-amino acid polymer are linked to the glycans of formula (I) via the carbonyl group of L1; and the remaining lysine side chains in the poly-lysine polymer are capped with 2,3- dihydroxypropylthioacetyl .
41. An anti-GMl antibody-binding multimeric compound of formula (III):
Figure imgf000080_0002
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group.
Z1 is -0-, -S-, -NR2- or -C(R2)2-, wherein each R2 is independently selected from H, (C1-C4)- alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5;
Ar is optionally substituted aryl or optionally substituted heteroaryl (e.g., monocyclic aryl or heteroaryl, or bicyclic aryl or heteroaryl);
L1 is a linker; m is at least 2;
P is a polymer; L2 is an optional linker; n is 2 to 6; and B is a branching moiety.
42. The compound of claim 41, wherein n is 2 whereby the compound is dimeric.
43. The compound of claim 41, wherein n is 3 whereby the compound is trimeric.
44. The compound of claim 42 or 43, wherein B is optionally substituted aryl or optionally substituted heteroaryl group substituted at two or more positions with L2 linkers.
45. The compound of claim 44, wherein B is optionally substituted phenyl.
46. The compound of claim 45, wherein B is
Figure imgf000081_0001
47. The compound of one of claims 41 to 46wherein each P is an a-amino acid polymer backbone, and each L2 links the C-terminal residue of the a-amino acid polymer backbone to a carbonyl group of B via amide bonds.
48. The compound of any one of claims 41 to 46, wherein each P is an a-amino acid polymer backbone, and each L2 links the N-terminal residue of the a-amino acid polymer backbone to B.
49. The compound of any one of claims 41 to 48, wherein each L2 is a linear linker comprising one or more linking moieties independently selected from -Ci-6-alkylene-, -NHCO-Ci-6-alkylene-, - CONH-C i-6-alkylene-, -0(CH2)p-, -(OCH2CH2)p-, -NHCO-, -CONH-, -NHS02-, -S02NH-, -CO- , — S02— , -0-, — S — , pyrrolidine-2, 5-dione, 1,2,3-triazolyl, -NH-, and -NMe-, wherein p is 1 to 10.
50. The compound of claim 49, wherein each L2 is -NH-(CH2)P-NH-, wherein p is 2 to 6.
51. The compound of any one of claims 41 to 50, wherein Ar is
Figure imgf000082_0001
wherein: q is 0 to 4; and each R11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
52. The compound of any one of claims 41 to 51, wherein:
R1 is selected from:
Figure imgf000082_0002
R21 is optionally substituted (Ci-C3)-alkyl (e.g. methyl or hydroxymethyl); and R22 and R23 are independently selected from H, optionally substituted (Ci-C3)-alkyl, optionally substituted aryl-(Ci-C3)-alkylene-, optionally substituted heteroaryl-(Ci-C3)-alkylene-, optionally substituted cycloalkyl-(Ci-C3)-alkylene-, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl.
53. The compound of claim 52, wherein R1 is
Figure imgf000082_0003
or a pharmaceutically acceptable salt thereof.
54. The compound of claim 52, wherein R1 is
Figure imgf000083_0001
or a pharmaceutically acceptable salt thereof.
55. The compound of claim 52, wherein R1 is a substituted carboxymethyl group selected from:
Figure imgf000083_0002
or a pharmaceutically acceptable salt thereof.
56. The compound of claim 55, wherein R1 is selected from:
Figure imgf000083_0003
or a pharmaceutically acceptable salt thereof.
57. The compound of any one of claims 41 to 56, wherein Z1 is -0-.
58. The compound of any one of claims 41 to 56, wherein Z1 is -S-.
59. The compound of any one of claims 41 to 56, wherein Z1 is -NR2-.
60 The compound of any one of claims 41 to 56, wherein Z1 is -NH-.
61. The compound of any one of claims 41 to 56, wherein Z1 is -C(R2)2-.
62. The compound of any one of claims 41 to 56, wherein Z1 is -CTf-.
63. The compound of any one of claims 41 to 62, wherein each L1 is linear linker comprising one or more linking moieties independently selected from -Ci-6-alkylene-, -NHCO-Ci-6-alkylene-, -
CONH-Ci-6-alkylene-, -NH Ci-6-alkylene-, -NHCONH-Ci-6-alkylene-, - NHCSNH-Ci-6-alkylene-, -Ci-6-alkylene-NHCO-, -Ci-6-alkylene-CONH-, -Ci-6-alkylene-NH-, -Ci-6-alkylene-NHCONH-, - C i-6-alkylene-NHC SNH-, -0(CH2)p-, -(OCH2CH2)p-, -NHCO-, -CONH-, -NHS02- -S02NH-, - CO-, — S02— , -0-, — S — , pyrrolidine-2,5 -dione, -NH-, and -NMe- wherein p is 1 to 10.
64. The compound of claim 63, wherein each L1 is -(CH2)2NH(C=0)(CH2)3S-CH2-(C=0)- connecting Ar to an amino-containing group of the repeat unit of a polymer backbone.
65. The compound of any one of claims 41 to 64, wherein each P is a polymer backbone having an average molecular weight in the range from 10 to 300 kDa.
66. The compound of any one of claims 41 to 65, wherein each P comprises a polypeptide.
67. The compound of claim 66, wherein each P comprises an a-amino acid polymer backbone having a mean length of 10-5000 amino acid residues (e.g., 20 to 2000, 30 to 1000, or 50 to 800 amino acid residues).
68. The compound of claim 67, wherein the a-amino acid polymer backbone has a mean length of 50 to 800 amino acid residues (e.g., 100, 150, 200, 300, 400, 600 or 800).
69. The compound of claim 68, wherein the a-amino acid polymer backbone has a mean length of 100 to 600 amino acid residues
70. The compound of any one of claims 67 to 69, wherein the a-amino acid polymer backbone has a mean length of at least 200 amino acid residues.
71. The compound of claim 70, wherein each P polymer has a mean length of 50 to 200 amino acid residues (e.g., 50, 100, or 200 amino acid residues).
72. The compound of claim 71, wherein the a-amino acid polymer backbone has a mean length of about 100 or 200 amino acid residues.
73. The compound of any one of claims 67 to 72, wherein each P polymer has a backbone consisting essentially of a-amino acid residues selected from lysine, ornithine, glutamic acid and aspartic acid.
74. The compound of claim 73, wherein each P polymer backbone is poly-lysine.
75. The compound of claim 74, wherein each P polymer is poly-Z-lysine.
76. The compound of claim 74, wherein each P polymer is poly-D-lysine.
77. The compound of claim 73, wherein each P polymer is poly-glutamic acid.
78. The compound of any one of claims 67 to 77, wherein 10% to 50% of the residues of each P polymer are linked to the glycans of formula (III).
79. The compound of claim 78, wherein 10% to 40% of the residues of each P polymer are linked to the glycans of formula (III).
80. The compound of claim 79, wherein 20% to 40% of the residues of each P polymer are linked to the glycans of formula (III).
81. The compound of claim 80, wherein 25% to 40% of the residues of each P polymer are linked to the glycans of formula (III).
82. The compound of any one of claims 78 to 81, wherein the loading of amino acid residues of each P polymer with the glycans of formula (III) is determined by NMR or quantitative NMR.
83. The compound of any one of claims 78 to 82, wherein each P polymer is poly-lysine and the remaining lysine side chains in the poly-lysine polymer are capped with a water solubilizing substituent.
84. The compound of claim 83, wherein the remaining lysine side chains in the poly-lysine polymer are capped with 2,3-dihydroxypropylthioacetyl.
85. The compound of any one of claims 41 to 84, wherein R1 is selected from:
Figure imgf000085_0001
86. The compound of claim 85, wherein the compound comprises a multitude of glycans of the following structure linked to each P:
Figure imgf000086_0001
or a pharmaceutically acceptable salt thereof.
87. The compound of claim 85, wherein the compound comprises a multitude of glycans of the following structure linked to each P:
Figure imgf000086_0002
or a pharmaceutically acceptable salt thereof.
88. The compound of any one of claims 86 to 87, wherein: each L1 is -(CH2)2NH(C=0)(CH2)3S-CH2-(C=0)-; each P is poly-lysine polymer backbone having a mean length of 50 to 200 amino acid residues (e.g., 50, 100 or 200);
25% to 40% of the lysine side chains in the a-amino acid polymer are linked to the multitude of glycans via the carbonyl group of L1; and the remaining lysine side chains in the poly-lysine polymer are capped with 2,3- dihydroxypropylthioacetyl .
89. A multimeric anti-GMl antibody-binding polymeric compound of formula (IV):
Figure imgf000086_0003
(IV) or a pharmaceutically acceptable salt thereof, wherein:
M1, M2 and M3 are each independently monomeric units that together provide a polymer backbone (P) having a mean length of 10-5000 amino acid residues (e.g., 20 to 2000, 30 to 1000, or 50 to 800 amino acid residues), x is 10 to 50 mol% of M1 units in the polymer backbone (P); y is 0 to 90 mol% of M2 units in the polymer backbone (P);
Y2 is an optional terminal group;
L1 is a linker that connects a glycan (G) to the M1 monomeric unit;
WSG is a water solubilizing group linked to the M2 monomeric unit;
L2 is an optional linker;
B is a branching moiety that covalently links to “n” polymer backbones, wherein n is 2 to 6; and
G is
Figure imgf000087_0001
wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group.
Z1 and Z2 are selected from -0-, -S-, -NR2- and -C(R2)2-, wherein each R2 is independently selected from H, (Ci-C -alkyl, (Ci-C- -alkoxy, -CH2C6H5, -CH2CH2C6H5, - OCH2C6H5, and -OCH2CH2C6H5; and
Ar is selected from optionally substituted aryl, optionally substituted heteroaryl.
90. The compound of claim 89, wherein n is 2 whereby the compound is dimeric.
91. The compound of claim 89, wherein n is 3 whereby the compound is trimeric.
92. The compound of claim 90 or 91, wherein B is optionally substituted aryl or optionally substituted heteroaryl group substituted at two or more positions with L2 linkers.
93. The compound of claim 92, wherein B is optionally substituted phenyl.
94. The compound of claim 93, wherein B is
Figure imgf000088_0001
95. The compound of claim 94, wherein: each L2 is -NH(CH2)PNH-, wherein p is 2 to 6, each M1, M2 and M3, if present, are amino acid residues, whereby P is an a-amino acid polymer backbone, wherein each L2 links the C-terminal of a P to a carbonyl group of B via amide bonds.
96. The compound of any one of claims 48 to 51, wherein each G-L1- is
Figure imgf000088_0002
or a pharmaceutically acceptable salt thereof.
97. The compound of any one of claims 48 to 51, wherein each G-L1- is
Figure imgf000089_0001
or a pharmaceutically acceptable salt thereof.
98. A compound of formula (V):
Figure imgf000089_0002
or a salt thereof, wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group.
Z1 is -0-, -S-, -NR2- or -C(R2)2-, wherein each R2 is independently selected from H, (C1-C4)- alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5;
Ar is optionally substituted aryl or optionally substituted heteroaryl (e.g., monocyclic aryl or heteroaryl, or bicyclic aryl or heteroaryl);
L1 is a linker;
Z2 is a chemoselective conjugation group.
99. The compound of claim 98, wherein Ar is
Figure imgf000090_0001
wherein: q is 0 to 4; and each R11 is independently selected from H, OH, optionally substituted (Ci-C3)-alkyl, optionally substituted (Ci-C3)-alkoxy, and halogen.
100. The compound of claim 98 or 99, wherein:
R1 is selected from:
Figure imgf000090_0002
R21 is optionally substituted (Ci-C3)-alkyl (e.g. methyl or hydroxymethyl); and R22 and R23 are independently selected from H, optionally substituted (Ci-C3)-alkyl, optionally substituted aryl-(Ci-C3)-alkylene-, optionally substituted heteroaryl-(Ci-C3)-alkylene-, optionally substituted cycloalkyl-(Ci-C3)-alkylene-, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted cycloalkyl.
101. The compound of claim 100, wherein R1 is
Figure imgf000090_0003
or a pharmaceutically acceptable salt thereof.
102. The compound of claim 100, wherein R1 is
Figure imgf000091_0001
or a pharmaceutically acceptable salt thereof.
103. The compound of claim 100, wherein R1 is a substituted carboxymethyl group selected from:
Figure imgf000091_0002
or a pharmaceutically acceptable salt thereof.
104. The compound of claim 103, wherein R1 is selected from:
Figure imgf000091_0003
or a pharmaceutically acceptable salt thereof.
105. The compound of any one of claims 98 to 104, wherein Z1 is -0-.
106. The compound of any one of claims 98 to 104, wherein Z1 is -S-.
107. The compound of any one of claims 98 to 104, wherein Z1 is -NR2-.
108. The compound of any one of claims 98 to 104, wherein Z1 is -NH-.
109. The compound of any one of claims 98 to 104, wherein Z1 is -C(R2)2-.
110. The compound of any one of claims 98 to 104, wherein Z1 is -CTf-.
111. The compound of any one of claims 98 to 110, wherein L1 is linear linker comprising one or more linking moieties independently selected from -Ci-6-alkylene-, -NHCO-Ci-6-alkylene-, -CONH- Ci-6-alkylene-, -NH Ci-6-alkylene-, -NHCONH-Ci-6-alkylene-, - NHCSNH-Ci-6-alkylene-, -Ci-6- alkylene-NHCO, -Ci-6-alkylene-C0NH-, -Ci-6-alkylene-NH-, -Ci-6-alkylene-NHC0NH-, -Ci-6- alkylene-NHC SNH-, -0(CH2)p- -(OCH2CH2)p-, -NHC0-, -C0NH-, -NHS02-, -S02NH- -C0-, — S02— , -0-, — S — , pyrrolidine-2,5 -dione, -NH-, and -NMe-, wherein p is 1 to 10.
112. The compound of any one of claims 98 to 110, wherein Z2 is selected from -NH2, -SH, -N3, alkyne, active ester, and maleimide.
113. The compound of claim 112, wherein -L'-Z2 is -(Ci-C6)-alkyl-NH2.
114. The compound of claim 113, wherein -L'-Z2 is -(CH2)2NH2.
115. The compound of claim 112, wherein -L'-Z2 is -(Ci-C6)-alkyl-amido-(Ci-C6)-alkyl-SH.
116. The compound of claim 115, wherein -L'-Z2 is -(CH2)2NHCO(CH2)3SH.
117. The compound of claim 116, wherein the compound is selected from:
Figure imgf000092_0001
or a salt thereof.
118. A pharmaceutical composition comprising : a compound according to any one of claims 1 to 97; and a pharmaceutically acceptable excipient.
119. A method of inhibiting or specifically binding an anti-GMl antibody in a sample, the method comprising contacting a sample comprising the anti-GMl antibody with an effective amount of the compound according to any one of claims 1 to 97.
120. The method of claim 119, wherein the anti-GMl antibody is an IgG anti-GMl antibody.
121. The method of claim 119, wherein the anti-GMl antibody is an IgM anti-GMl antibody.
122. The method of any one of claims 119 to 121, wherein the sample is a biological sample.
123. The method of claim 122, wherein the biological sample is a blood sample.
124. The method of claim 122 or 123, wherein the biological sample is contacted ex vivo.
125. The method of any one of claims 119 to 124, further comprising detecting the anti-GMl autoantibody that specifically binds to the compound.
126. The method of claim 125, further comprising determining the level of the anti-GMl autoantibody in the sample.
127. The method of any one of claims 119 to 126, further comprising obtaining the sample from a subject having, or suspected of having a neurological condition associated with the anti-GMl antibody.
128. An anti-GMl antibody-binding compound according to any one of claims 1 to 97 for use in diagnosis of a neurological condition associated with an anti-GMl autoantibody in a human subject.
129. Use of an anti-GMl antibody-binding compound according to any one of claims 1 to 97 in the manufacture of a medicament for diagnosis of a neurological condition associated with an anti-GMl autoantibody in a human subject.
130. A method of alleviating one or more symptoms of a neurological condition associated with an anti-GMl autoantibody, the method comprising administering to a human subject having the neurological condition a therapeutically effective amount of the pharmaceutical composition according to claim 98, or the compound according to any one of claims 1 to 97.
131. The method of claim 130, wherein the anti-GMl autoantibody is an IgG anti-GMl antibody.
132. The method of claim 130, wherein the anti-GMl autoantibody is an IgM anti-GMl antibody.
133. The method of any one of claims 130 to 132, wherein the neurological condition is anti-GMl antibody mediated neuropathy.
134. The method of claim 130, further comprising, prior to the administration, determining the type and/or level of an anti-GMl autoantibody in a biological sample of the subject.
135. The method of any one of claims 130 to 134, wherein the neurological condition is multifocal motor neuropathy (MMN).
136. The method of any one of claims 130 to 134, wherein the neurological condition is Guillain- Barre Syndrome (GBS).
137. The method of claim 136, wherein the neurological condition is selected from acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) and the pharyngeal-cervical -brachial variant of GBS.
138. An anti-GMl antibody-binding compound according to any one of claims 1 to 97, or a pharmaceutical composition according to claim 98, for use in alleviating one or more symptoms of a neurological condition associated with an anti-GMl autoantibody in a human subject.
139. Use of an anti-GMl antibody-binding compound according to any one of claims 1 to 97, or a pharmaceutical composition according to claim 98, in the manufacture of a medicament for use in alleviating one or more symptoms of a neurological condition associated with an anti-GMl autoantibody in a human subject.
140. A method of treating anti-GMl antibody mediated neuropathy, the method comprising administering to a human subject having the neuropathy a therapeutically effective amount of the pharmaceutical composition of claim 98, or the compound according to any one of claims 1 to 97.
141. The method of claim 140, wherein the neuropathy is multifocal motor neuropathy (MMN).
142. The method of claim 140, wherein the neuropathy is Guillain-Barre Syndrome (GBS).
143. The method of claim 142, wherein the neuropathy is selected from acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) and the pharyngeal-cervical -brachial variant of GBS.
144. An anti-GMl antibody-binding compound according to any one of claims 1 to 97, or a pharmaceutical composition according to claim 98, for use in treating a neurological condition associated with an anti-GMl autoantibody in a human subject.
145. Use of an anti-GMl antibody-binding compound according to any one of claims 1 to 97, or a pharmaceutical composition according to claim 98, in the manufacture of a medicament for use in treating a neurological condition associated with an anti-GMl autoantibody in a human subject.
146. A bead composition for binding anti-GMl autoantibodies, comprising: a plurality of beads; and an anti-GMl antibody-binding compound of formula (I):
Figure imgf000095_0001
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a sialic acid group or an optionally substituted carboxymethyl group;
Z1 is -0-, -S-, -NR2- or -C(R2)2-, wherein each R2 is independently selected from H, (C1-C4)- alkyl, (Ci-C4)-alkoxy, -CH2C6H5, -CH2CH2C6H5, -OCH2C6H5, and -OCH2CH2C6H5;
Ar is optionally substituted aryl or optionally substituted heteroaryl;
L1 is a linker; m is at least 2; and
Y is a bead of the plurality of beads.
147. The bead composition of claim 146, wherein the bead is an agarose, a sepharose, a dextran, a cellulose, chitin, chitosan and the derivative of the above-mentioned substance, an organic or inorganic porous material, a magnetic bead, or a micro bead.
148. The bead composition of claim 146, wherein Y is a sepharose or agarose bead.
149. The bead composition of claim 146, wherein the anti-GMl antibody-binding compound of formula (I) is as described in any one of claims 2 to 15.
150. An immune -adsorption apparatus for removing anti-GMl autoantibodies from a biological sample of a subject in need thereof, comprising the bead composition according to any one of claims 1 to 149.
151. A method of ex vivo treatment of a neurological condition associated with an anti-GMl autoantibody, the method comprising
(a) obtaining a biological sample (e.g., peripheral blood) from a human patient having the neurological condition;
(b) contacting the biological sample with an amount of the compound according to any one of claims 1-97, the bead composition according to any one of claims 146-149, or the apparatus according to claim 150, effective to remove substantially all anti-GMl autoantibody present in the biological sample;
(c) administering the contacted biological sample lacking the anti-GMl autoantibody to the human patient.
152. The method of claim 151, wherein the anti-GMl autoantibody is an IgG anti-GMl antibody.
153. The method of claim 151, wherein the anti-GMl autoantibody is an IgM anti-GMl antibody.
154. The method of any one of claims 151 to 153, wherein the neurological condition is anti-GMl antibody mediated neuropathy.
155. The method of any one of claims 151 to 154, wherein the compound is immobilized on a support.
156. The method of claim 155, wherein the support is a bead composition.
157. The method of claim 156, wherein the bead composition comprises magnetic beads, and the contacting step further comprises application of a magnetic field to separate the beads and from the contacted biological sample.
158. The method of claim 156, wherein the bead composition is comprised in a column or cartridge and the contacting step further comprises flowing the contacted biological sample through the column or cartridge to collect the contacted biological sample.
159. The method of any one of claims 151 to 158, wherein the neurological condition is multifocal motor neuropathy (MMN).
160. The method of any one of claims 151 to 158, wherein the neurological condition is Guillain- Barre Syndrome (GBS).
161. The method of claim 151, wherein the neurological condition is selected from acute motor axonal neuropathy (AMAN), acute motor-sensory axonal neuropathy (AMSAN), acute inflammatory demyelinating polyneuropathy (AIDP) and the pharyngeal-cervical -brachial variant of GBS.
162. Use of an anti-GMl antibody-binding compound according to any one of claims 1 to 97, in the manufacture of a medicament for use in ex vivo treatment of a neurological condition associated with an anti-GMl autoantibody in a human subject.
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